Compositions for FNIP1/FNIP2 Gene Modulation and Methods Thereof

ABSTRACT

Compositions, systems, and methods are described herein to modulate (reduce/inhibit) expression/activity of FNIP1 and/or FNIP2 (“FNIP1/FNIP2”) in a cell, animal or human subject, which can prevent, ameliorate, or treat diseases (neuromuscular or neurodegenerative diseases). Methods are described for modulating (reducing/inhibiting) FNIP1/FNIP2 expression/activity via a modulator to regulate FNIP1/FNIP2 expression/activity. Also included are compositions containing modulators that regulate FNIP1/FNIP2 expression/activity. Pharmaceutical compositions, kits, and methods of delivering compositions used in modulating, reducing, or inhibiting FNIP1/FNIP2 expression/activity are described. Methods to develop, synthesize, or produce modulators, and treat ALS and related disorders (TDP-43 proteinopathies, oxidative stress, obesity, anemia, or ischemic diseases) are provided. Also provided are compositions, systems, and methods to modulate (increase) FNIP1/FNIP2 expression/activity in a cell, an animal or human subject, which can treat, prevent, or ameliorate diseases. Furthermore, described are diagnostic and testing methods to detect FNIP1/FNIP2 associated variants or expression/activity levels, and compositions comprising diagnostic or testing kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 63/284,187 (filed on Nov. 30, 2021), the disclosure of which is incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The instant application contains a Sequence Listing, which has been filed electronically. The contents of the electronic sequence listing (071528_P4US3.xml; Size: 2,170,000 bytes; and Date of Creation: Nov. 29, 2022) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions, systems, and methods for modulating, in particular reducing or inhibiting, the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal or human subject. Such compositions, systems, and methods can be useful to treat, prevent, or ameliorate diseases, particularly neuromuscular or neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AIMD), and other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia and ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. This invention also relates to compositions, systems, and methods for modulating, in particular increasing, the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal or human subject, which can be useful to treat, prevent, or ameliorate diseases, particularly inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers such as fibrofolliculomas and kidney tumors.

BACKGROUND

In the following discussion certain articles and processes will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and processes referenced herein do not constitute prior art under the applicable statutory provisions.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is characterized by the progressive degeneration and loss of survival of both upper motor neurons in the brain and lower motor neurons in the spinal cord. This results in a lack of muscle stimulation, leading to muscle atrophy and fasciculations. Around 10% of ALS patients can survive for 10 or more years. However, most patients (around 90%) ultimately die from respiratory failure within 3 to 5 years from the onset of symptoms. ALS affects over 30,000 in the US alone, with over 5,600 new cases annually. There is currently a lack of accurate diagnostics and effective prophylaxis or therapies for ALS (Khairoalsindi and Abuzinadah, Neurology Research International, Article ID 6534150 (2018)). The drug Riluzole, which was approved by the FDA in 1995, can in most cases only extend the survival of ALS patients by 2-3 months. Edaravone (Radicava), which was approved by the FDA in 2017, slows the decline in physical function in ALS patients but is only effective for a subset of patients (around 7%), and long-term data on any survival benefit is still lacking. The lack of a long-term, effective therapy for ALS highlights the urgent need for the development of novel ALS therapies.

It is important to identify the genetic features in patients that have ALS, as an understanding of the molecular basis of the disease will aid the development of effective modulators that can be used as prophylactics or therapeutics to prevent or treat the disease, respectively. ALS is a complex disease that arises from the interplay of multiple genetic factors that are still poorly understood. 90-95% of ALS cases are sporadic, occurring apparently at random in individuals without a family history of ALS. In contrast, about 5-10% of ALS cases are familial, occurring in individuals with a family history of ALS (Renton et al., Nature Neuroscience, 17(1): 17-23 (2014)). To date, studies of familial and sporadic ALS cases have uncovered mutations in over 30 genes (e.g., C9orf72, SOD1, STMN2, NEK1, TARDBP, FUS, VCP, OPTN, SQSTM1, UBQLN2, hnRNPA1, MATR3, and others) that collectively account for less than 17% of all ALS cases (Renton et al., Nature Neuroscience, 17(1): 17-23 (2014)). However, the majority (>83%) of ALS cases have unknown causes.

In 2006, TAR DNA-binding protein 43 (TDP-43) was discovered to be a key component of insoluble and highly ubiquitinated aggregates in the brains of patients suffering from ALS and frontotemporal lobar dementia (FTLD). Despite the potentially diverse genetic etiology of ALS, strikingly, over 97% of all ALS cases (both sporadic and familial) and around 45% of frontotemporal lobar degeneration (FTLD; also called frontotemporal dementia (FTD) which is a type of FTLD) cases display TDP-43 positive aggregates in the cytoplasm of affected neurons (Prasad et al., Frontiers in Molecular Neuroscience, 12:25 (2019)). The TDP-43 present in aggregates can be unmodified TDP-43 or modified forms of TDP-43, such as phosphorylated TDP-43 (pTDP-43), ubiquitinated TDP-43 or truncated TDP-43. Thus, as used herein, TDP-43 can refer to unmodified and/or modified forms of TDP-43 including phosphorylated, ubiquitinated or truncated forms of TDP-43. In particular, phosphorylation of TDP-43 at Ser-379, Ser-403, Ser-404, and especially Ser-409/Ser-410, is a key signature of TDP-43 proteinopathies, including ALS (Neumann et al., Acta Neuropathol., 117(2):137-149 (2009)). Furthermore, mutations in TARDBP, the gene that encodes for TDP-43, have been associated with familial cases of ALS, thus cementing its central role in ALS. In general, once initiated, the aggregation of TDP-43 in the cytoplasm can proceed in a self-propagating manner, involving polymerization of RNA and protein molecules to form toxic products that are resistant to proteolysis. In healthy cells, TDP-43 is predominantly localized to the nucleus and carries out multiple important RNA processing functions there, including regulating RNA transcription, RNA splicing, RNA transport, and stability. Its multi-domain structure allows it to be a central modulator of multiple processes. For example, it comprises two RNA recognition motifs (RRM1 and RRM2) that mediate interactions with RNA and DNA, a C-terminal glycine-rich domain that mediates interactions with other proteins, as well as a nuclear localization signal (NLS) and nuclear export signal (NES) that regulates its shuttling between the nucleus and cytoplasm. Thus, TDP-43's involvement in ALS pathogenesis can be caused by a toxic gain-of-function mechanism due to increased levels of pathological TDP-43 aggregates in the cytoplasm, or a loss-of-function mechanism due to a decrease of functional TDP-43 levels in the nucleus, or a combination of both. The role of TDP-43 in ALS is described by Prasad et al. (Prasad et al., Frontiers in Molecular Neuroscience, 12:25 (2019)) and Scotter et al. (Scotter et al., Neurotherapeutics, 12(2): 352-363 (2015)), the disclosures of which, along with their references, are incorporated herein in its entirety.

Although insoluble cytoplasmic TDP-43 aggregates are the hallmark of ALS, there is evidence to suggest that biomolecular condensates of TDP-43, which are transient, liquid-like droplets of TDP-43, are the neurotoxic species (Gasset-Rosa et al., Neuron 102(2): 339-357 (2019)). These condensates are formed by the prion-like, low-complexity intrinsically disordered region (IDR) in the C-terminal domain of TDP-43, which allows it to undergo liquid-liquid phase separation (LLPS) in the cell. In fact, the formation of insoluble cytoplasmic TDP-43 aggregates has been suggested to be a defense mechanism of the cell to sequester TDP-43, to reduce the formation of toxic TDP-43 biomolecular condensates in the cytoplasm. As used herein, the term TDP-43 aggregates can refer to biomolecular condensates of TDP-43, as well as to any aggregates of TDP-43, whether soluble or not.

Consequently, a decrease in the levels of pathological TDP-43 aggregates in the cytoplasm, or an increase in the levels of normal TDP-43 in the nucleus, or a combination of both, are likely to be effective to treat, ameliorate, or prevent ALS or represent an indication thereof as a biomarker. Likewise, decreasing TDP-43 aggregates in the cytoplasm are likely to be effective to treat, ameliorate, or prevent other diseases, particularly neuromuscular or neurodegenerative diseases, involving either an increase in levels of pathological TDP-43 aggregates in the cytoplasm, or a decrease of functional TDP-43 levels in the nucleus, or a combination thereof. Such diseases are termed TDP-43 proteinopathies, examples of which include but are not limited to, ALS, Alzheimer's disease, argyrophilic grain disease, vascular dementia, frontotemporal dementia (FTD, FTD-TDP-43, and FTD-tau) and the greater group of frontotemporal lobar degeneration (FTLD), semantic dementia, dementia with Lewy bodies, polyglutamine diseases, Huntington's disease, spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, parkinsonism, Parkinson's disease (PD), Perry syndrome, ALS-parkinsonism dementia complex of Guam, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), pseudobulbar palsy, Mills' syndrome, monomelic amyotrophy, post-polio syndrome (PPS), madras motor neuron disease (MMND), progressive muscular atrophy (PMA), spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), retinal degeneration diseases such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), and glaucoma, wherein in each case at least a sub-population of patients can exhibit an increase in levels of TDP-43 aggregates in the cytoplasm. TDP-43 proteinopathies are further described in Gendron and Josephs (Gendron and Josephs, Neuropathol. Appl. Neurobiol. 36:97-112 (2010)), Lagier-Tourenne et al. (Lagier-Tourenne et al., Hum. Mol. Gen. 19(1):R46-R64 (2010)), and Matsukawa et al. (Matsukawa et al., Journal of Biological Chemistry, 291(45): 23464-23476 (2016)), the disclosures of which, together with references cited therein, are incorporated herein in their entirety.

SUMMARY

Compositions, systems, and methods are described herein for the modulation, and in particular the reduction or inhibition, of expression of the FNIP1 and/or FNIP2 gene or the activity of FNIP1 and/or FNIP2 protein in a cell, animal or human subject, which can be used to prevent, ameliorate, or treat diseases, particularly neuromuscular or neurodegenerative diseases, such as ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), or other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. Also described herein are compositions, systems and methods for modulating, in particular increasing, the expression and/or activity of FNIP1 and/or FNIP2 gene or protein in a cell, animal or human subject, which can be used to treat, prevent or ameliorate diseases, particularly inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers, and other diseases that are linked to loss-of-function of FNIP1 and/or FNIP2. Methods are described for the modulation of FNIP1 and/or FNIP2 expression or activity, comprising the use of a modulator to increase or decrease FNIP1 and/or FNIP2 expression or activity. Included also herein are compositions for modulators used to increase or decrease FNIP1 and/or FNIP2 expression or activity. Related pharmaceutical compositions, kits, and methods of delivery of compositions used in modulating FNIP1 and/or FNIP2 expression or activity are also described. Methods are also described for the development, synthesis, and production of modulators, as well as for therapeutic treatment of diseases, such as ALS and other related disorders such as other TDP-43 proteinopathies, oxidative stress, obesity, anemia and ischemic diseases; as well as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, and cancers, such as fibrofolliculomas and kidney tumors. Furthermore, methods for diagnostics and testing comprising detecting FNIP1 and/or FNIP2 associated variants, or FNIP1 and/or FNIP2 expression or activity levels, as well as compositions comprising kits for diagnostics and testing, are described herein.

In some embodiments, provided herein are antisense modulators to modulate FNIP1 and/or FNIP2 expression or activity. In some embodiments, provided herein are antisense modulators to inhibit or reduce FNIP1 and/or FNIP2 expression or activity. In other embodiments, provided herein are antisense modulators to increase FNIP1 and/or FNIP2 expression or activity. In one embodiment, antisense modulators comprise antisense oligonucleotides (ASOs). In one embodiment, the ASO is between 12-30 nucleobases in length. In certain embodiments, the ASO is at least 70% complementary, alternatively at least 80% complementary, alternatively at least 85% complementary, alternatively at least 90% complementary, alternatively at least 95% complementary, alternatively at least 98% complementary, alternatively 100% complementary to an equal length portion of at least one target sequence described in SEQ ID NOs: 1-15. In certain embodiments, the ASO is at least 70% identical, alternatively at least 80% identical, alternatively at least 85% identical, alternatively at least 90% identical, alternatively at least 95% identical, alternatively at least 98% identical, alternatively 100% identical to an equal length portion of at least one sequence described in SEQ ID NOs: 16-1494. In one embodiment, the ASO includes at least one modification to an internucleoside linkage, a sugar, or a nucleobase component. In one embodiment, all of the internucleoside linkages of the ASO comprise phosphorothioate modifications. In another embodiment, all of the sugar components of the ASO comprise the 2′-MOE modification. In another embodiment, the ASO comprises a gapped sequence consisting of a central sequence of oligonucleotides without sugar modifications, which are flanked on both sides by wing sequences consisting of 2′-MOE modified nucleotides. In another embodiment, the ASO comprises a gapped sequence consisting of a central sequence of deoxynucleotides wherein the second nucleotide of the central sequence from the 5′ end of the ASO contains a 2′-OMe sugar modification, and which gapped sequence is flanked on both sides by wing sequences consisting of 2′-MOE modified nucleotides. In one embodiment, all cytosine nucleobases of the ASO comprise 5-methylcytosine modifications. In certain embodiments, the ASO is conjugated to one or more molecules, such as a peptide or polypeptide, lipid, sugar, nucleotide or oligonucleotide, other polymer, cleavage agent, transport agent, intercalating agent, molecular beacon, hybridization-triggered crosslinking agent, lipophilic agent, or hydrophilic agent. In one embodiment, the ASO is conjugated to one or more N-acetyl galactosamine (GalNAc) residue or other such conjugates or complexes.

In some embodiments, antisense modulators comprise modulators used in RNA interference (RNAi), such as siRNAs, miRNAs and shRNAs. In one embodiment, the antisense modulator is a siRNA. In one embodiment, the antisense region of the siRNA is 19 to 29 nucleotides in length. In certain embodiments, the antisense region of the siRNA is at least 70% complementary, alternatively at least 80% complementary, alternatively at least 85% complementary, alternatively at least 90% complementary, alternatively at least 95% complementary, alternatively at least 98% complementary, alternatively 100% complementary to an equal length portion of at least one target sequence described in SEQ ID NOs: 1-15. In certain embodiments, the antisense region of the siRNA is at least 70% identical, alternatively at least 80% identical, alternatively at least 85% identical, alternatively at least 90% identical, alternatively at least 95% identical, alternatively at least 98% identical, alternatively 100% identical to an equal length portion of at least one sequence described in SEQ ID NOs: 16-1494, wherein thymine is replaced by uracil. In one embodiment, the first two nucleotides at the 5′ end of the sense strand, as well as the first two nucleotides at the 5′ end of the antisense strand, of the siRNA are modified with a 2′-O-alkyl group, such as a 2′-OMe group.

In other embodiments, provided herein are modulators other than antisense modulators, for example other oligonucleotide modulators (e.g., ribozyme, deoxyribozyme, or aptamers), antibody modulators, peptide modulators, small molecule modulators, and nucleic acid vectors, for modulating the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal or human subject. In some embodiments, provided herein are modulators for gene therapy. In one embodiment, the modulator for gene therapy comprises a nucleic acid vector encoding one or more functional copies of FNIP1 and/or FNIP2, as described in SEQ ID NOs: 1-15, in order to increase the levels of FNIP1 and/or FNIP2 in the cell. In another embodiment, the nucleic acid vector is modified to enable viral delivery. In other embodiments the nucleic acid vector is modified to enable non-viral delivery. In certain embodiments, provided herein are antibody modulators that are chosen from the set of modulators described in Table 14. In some embodiments, the antibody, antibody fragment, monobody or peptide modulator binds to the same epitope as at least one antibody modulator described in Table 14. In other embodiments, the antibody, antibody fragment, monobody or peptide modulator binds to a different epitope to that of the modulators described in Table 14. In some embodiments, the antibody, antibody fragment, monobody, or peptide modulator comprises a complementarity-determining region (CDR) that is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the CDR of at least one antibody modulator described in Table 14, as assessed by sequence alignment or other scoring methods known in the art. In certain embodiments, provided herein are small molecule modulators comprising at least one exemplar small molecule modulator described in Table 12 and/or Table 13. In certain embodiments, the small molecule modulator comprises at least one scaffold described in Table 12 and/or Table 13. In some embodiments, the small molecule modulators, or part thereof, have a Tanimoto index of at least 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00 compared to at least one exemplar or scaffold described in Table 12 and/or Table 13.

In certain embodiments, provided herein are methods and compositions for the delivery of antisense modulators into a cell, an animal, or a human subject, in order to modulate the expression or activity of FNIP1 and/or FNIP2. In one embodiment, delivery methods and compositions comprise a transfection reagent, such as a liposomal-based or amine-based transfection reagent. In one embodiment, the antisense modulators are delivered naked without a transfection reagent. In one embodiment, the antisense modulators are delivered via a nucleic acid vector. In one embodiment, the method of delivery includes one or more of the common delivery methods used to deliver drugs, such as intrathecal injection.

One set of embodiments provide for pharmaceutical compositions, comprising a modulator and a pharmaceutically acceptable carrier or diluent, which can be administered to a cell, an animal, or a human subject to modulate, and in particular to reduce or inhibit, the expression or activity of FNIP1 and/or FNIP2 in the cell, animal or human subject. Another set of embodiments provide for pharmaceutical compositions, comprising a modulator and a pharmaceutically acceptable carrier or diluent, which can be administered to a cell, an animal, or a human subject to modulate, and in particular to increase, the expression or activity of FNIP1 and/or FNIP2 in the cell, animal or human subject. In one embodiment, the pharmaceutical composition is administered intrathecally. Such treatment methods can be used to treat, ameliorate, or prevent ALS and other diseases, such as other TDP-43 proteinopathies, oxidative stress, obesity, anemia, or ischemic diseases; as well as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, and cancers. In one embodiment, a pharmaceutical composition described herein is co-administered with one or more other pharmaceutical agents, such as for example, Riluzole (Rilutek), Dexpramipexole, Edaravone, Tofersen, Baclofen (Lioresal), or other drug that is typically administered to treat, ameliorate, or manage symptoms in ALS. In another embodiment, a pharmaceutical composition described herein is co-administered with one or more pharmaceutical agents or other drug that is typically administered to treat, ameliorate, or manage symptoms in oxidative stress, obesity, anemia or ischemic diseases; as well as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, and cancers.

Some embodiments provide for the testing and monitoring of FNIP1 and/or FNIP2 levels or activity in a cell, an animal, or a human subject. In some embodiments, these tests can be used for the purposes of diagnosing a disease such as ALS, other TDP-43 proteinopathy, oxidative stress, obesity, anemia, or ischemic disease; as well as inflammatory disease, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, and cancer. In other embodiments, these tests can be used for the purposes of determining risk or susceptibility to said diseases. In some embodiments, these tests can be used for the purposes of monitoring the progression or response to a treatment for said diseases. In other embodiments, provided herein are methods of determining risk or susceptibility, methods of diagnosis, methods of predicting prognosis, or methods of assessing a human individual for a probability of a response to a therapeutic method and/or modulator for neuromuscular or neurodegenerative diseases, such as, for example, FTLD, Alzheimer's Disease, retinal degeneration diseases such as age-related macular degeneration (AMD), and other TDP-43 proteinopathies disclosed herein. In other embodiments, provided herein are methods of determining risk or susceptibility, methods of diagnosis, methods of predicting prognosis, or methods of assessing a human individual for a probability of a response to a therapeutic method and/or modulator for oxidative stress, obesity, anemia, or ischemic disease; as well as inflammatory disease, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, and cancer.

DETAILED DESCRIPTION

The following description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. Various modifications to the example embodiments and the genetic principles and features described herein will be readily apparent. The example embodiments are mainly described in terms of particular processes and systems provided in particular implementations. However, the processes and systems will operate effectively in other implementations. Phrases such as “example embodiment”, “one embodiment”, and “another embodiment” can refer to the same or different embodiments.

The example embodiments will be described with respect to methods and compositions having certain components. However, the methods and compositions can include more or less components than those shown, and variations in the arrangement and type of the components can be made without departing from the scope of the invention.

The example embodiments will also be described in the context of methods having certain steps. However, the methods and compositions operate effectively with additional steps and steps in different orders that are not inconsistent with the example embodiments. Thus, the present invention is not intended to be limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and features described herein and as limited only by appended claims.

It should be noted that as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to the effect of “a neuron” can refer to the effect of one or a combination of neurons, and reference to “a method” includes reference to equivalent steps and processes known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range—and any other stated or intervening value in that stated range—is encompassed within the invention. Where the stated range includes upper and lower limits, ranges excluding either of those limits are also included in the invention.

Unless expressly stated, the terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the formulations and processes that are described in the publication and which might be used in connection with the presently described invention.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein in the detailed description and figures. Such equivalents are intended to be encompassed by the claims.

For simplicity, in the present document certain embodiments are described with respect to use of certain methods. It will become apparent to one skilled in the art upon reading this disclosure that the invention is not intended to be limited to a specific use, and can be used in a wide array of implementations.

Throughout this specification, the oligonucleotides and polypeptides referred to include all enantiomers, stereoisomers, racemic mixtures, optically pure isomer forms, complementary sequences, modified or analog forms, and both deoxyribonucleotide (or DNA) and ribonucleotide (or RNA) forms.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the embodiments pertain.

“Nucleic acid sequence data” as used herein refers to any sequence data obtained from nucleic acids from an individual. Such data includes, but is not limited to, deoxyribonucleotide (DNA) data, ribonucleotide (RNA) data, whole genome sequencing data, exome sequencing data, genotyping data, transcriptome sequencing data, complementary DNA or cDNA library sequencing data, and the like. Nucleic acid sequences are written in the 5′ to 3′ direction.

“Nucleobases”, or “bases”, are used interchangeably and refer to nitrogen-containing compounds that form nucleosides, which in turn are components of nucleotides. The five primary or natural nucleobases are adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). Other nucleobases, such as synthetic or modified nucleobases, are included herein and detailed below.

“Nucleotide” refers to a compound comprising a nucleoside and a linkage group, commonly a phosphate linkage group. Nucleotides include both natural and modified nucleotides.

“Sugar” or “sugar component” can mean a natural or modified sugar, which includes ribose sugars and deoxyribose sugars found in RNA and DNA, respectively, as well as other modified sugars detailed below.

“Nucleoside” refers to a compound comprising both a nucleobase and sugar component. Nucleosides can be natural or modified.

“Nucleoside linkage” or “internucleoside” linkage refers to the covalent linkages of adjacent nucleosides. Nucleoside linkages comprise the primary linkages between nucleotides in an oligonucleotide.

“Chimeric compound” refers to a compound, most commonly an oligonucleotide, that comprises at least one nucleotide having at least one nucleobase, nucleoside linkage, or sugar component that differs from at least one other nucleotide within the same compound. This difference can originate from variations in how components of nucleotides within the same compound are modified or in some cases left unmodified. In some embodiments, similar or identical modifications to nucleotides in chimeric compounds can be grouped together spatially in regions. Any modification or combination of modifications described herein or elsewhere, including those modifications known to persons skilled in the art, can be included in a chimeric compound.

“Motif” refers to a region or subsequence within the sequence of an oligonucleotide, or polypeptide, that has a specific functional or biological significance. Examples of motifs include nucleobase sequences within an oligonucleotide, such as DNA or RNA, which are recognized by a DNA or RNA-binding protein. Other examples of motifs include amino acid sequences within a polypeptide that is responsible for a specific function of the polypeptide.

“Nucleic acid sequence”, “nucleobase sequence”, “nucleotide sequence”, or simply “sequence” are used interchangeably and refer to the sequence of nucleobases on a nucleic acid molecule or oligonucleotide.

“Coding DNA” refers to a DNA sequence that is transcribed to messenger RNA (mRNA) and subsequently translated to a polypeptide or protein.

“Non-coding DNA” refers to a DNA sequence that does not encode a polypeptide, including, but not limited to, a DNA sequence that is transcribed to a functional RNA (e.g., non-coding RNA (ncRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), regulatory RNA, microRNA (miRNA), small interfering RNA (siRNA), Piwi interacting RNA (piRNA) or long noncoding RNA (lncRNA)); a DNA sequence that contains a regulatory element such as a promoter, enhancer, terminator, insulator or silencer that affects the expression of one or more genes; a DNA sequence that performs a structural function (e.g., centromere, telomere, satellite); a DNA sequence that serves as a replication origin; a DNA sequence that is located within a protein-coding gene but is removed before a protein is made, otherwise known as an intron; or otherwise any DNA sequence with unknown function.

The term “mRNA” refers to messenger RNA, the message derived by the transcription of coding DNA to form precursor mRNA (pre-mRNA). Pre-mRNA is subsequently processed into mature mRNA by splicing to remove introns, and addition of a 5′ cap and poly-A tail. Mature mRNA is used as a template by ribosomes for translation into polypeptides. The term “mRNA” as used herein includes pre-mRNA, sometimes also referred to as hnRNA (heterogeneous nuclear RNA), mature mRNA, as well as mRNA in any stage of processing.

The term “gene” as used herein, refers to a DNA sequence that is transcribed to mRNA and subsequently translated to a polypeptide, and/or a DNA sequence that is transcribed to a functional RNA.

“Polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably, and refer to a polymer of two or more amino acids.

“Oligonucleotide” refers to a polymer comprising two or more nucleotides, and can also refer to a modified oligonucleotide. “Modified oligonucleotide” refers to an oligonucleotide comprising at least one modification to a nucleobase, nucleoside linkage, or sugar component.

An “allele”, also referred to as a “variant”, or “polymorphism”, refers to one of at least two different nucleotide sequence variations at a given position (locus) in the genome. Thus, a specific allele of a polymorphic site refers to a specific version of the sequence with respect to a polymorphic site. A “variant” or “polymorphism” can also refer to a specific allele of a polymorphic site that differs from a reference genome.

“Polymorphic marker”, also referred to as “polymorphic site” or simply as “marker”, refers to a genomic site with at least two sequence variants, or at least two alleles. Thus, genetic association with a polymorphic marker, refers to association with at least one specific allele of that polymorphic marker. “A marker” can also refer to a specific allele of a polymorphic marker. A polymorphic marker can refer to any type of sequence variation found in the genome, including but not limited to single nucleotide polymorphisms (SNPs), curated SNPs (cSNPs), insertions, deletions, copy number variations (CNVs), codon expansions, methylation status, translocations, duplications, repeat expansions, rearrangements, multi-base polymorphisms, splice variants, microsatellite polymorphisms etc. A “marker” can also refer to a “biomarker”.

A “single nucleotide polymorphism” or “SNP” is a type of variation of DNA where a single nucleotide at a specific location in a genome differs between two or more individuals, or two or more populations. Most SNPs have two alleles; in such cases, an individual is either homozygous for one allele at the polymorphic site, or heterozygous for both alleles.

An “insertion” or “deletion” is a variant with additional nucleotides or fewer nucleotides respectively compared to a reference DNA sequence.

A “microsatellite” is a type of polymorphic marker where there are multiple small repeats of bases that are 2-8 nucleotides in length.

A “haplotype” refers to a segment of genomic DNA containing a specific combination of alleles along the segment that tends to be inherited together in human evolution.

“Linkage disequilibrium” refers to the non-random association of alleles at different loci in a given population.

The term “associated with” refers to and can be used interchangeably with “within”, or “correlated with”, or “in linkage disequilibrium with”, or “functionally related with”, or any combination of the terms.

“Susceptibility” refers to the tendency, propensity or risk of an individual to develop a particular phenotype (e.g., a trait or a disease), or to being more or less able to resist developing a particular phenotype. The term encompasses decreased susceptibility to, or decreased risk of, or a protection against a disease. The term also encompasses an increased susceptibility to, or increased risk of developing, a disease.

The term “and/or” indicates “one or the other or both”. In other words, the term indicates that both or either of the items are involved.

The term “biomarker” refers to a biological molecule such as a protein, a polypeptide, a small molecule, a metabolite or a nucleic acid sequence that is associated with a phenotype such as a disease, and whose measurement can be used for determining a susceptibility to the disease, or prognosis for the disease, or diagnosis for the disease, or determining a response to a therapy for the disease.

The term “look-up table” is a table that links one form of data to another, or one or more forms of data to a predicted outcome (e.g., a trait, a disease, or other phenotype). Look-up tables can contain information about one or more polymorphic markers, one or more alleles at each polymorphic marker, and a correlation between alleles for a polymorphic marker and a particular phenotype (e.g., a trait or a disease).

A “computer-readable medium” is a medium for storage of information that is accessible by a computer interface that is custom-built or available commercially. Some examples of computer-readable media include, but are not limited to, optical storage media, magnetic storage media, memory, punch cards, or other commercially available media.

A “nucleic acid sample” refers to a DNA and/or RNA sample obtained from an individual. In certain embodiments (e.g., in detecting specific polymorphic markers and/or haplotypes), the nucleic acid sample comprises genomic DNA. Genomic DNA samples can be obtained from any source that contains genomic DNA, such as blood, saliva, tissue sample, cerebrospinal fluid, amniotic fluid etc.

A “sample” in general refers to any sample, such as a biological sample, obtained from an individual.

A “subject”, a “patient” or an “individual” refers to a living multi-cellular vertebrate organism, which includes both human and non-human mammals, unless otherwise indicated.

The term “therapeutic agent” refers to an agent that can be used for preventing, treating, or ameliorating symptoms associated with a disease.

The term “response to a therapeutic method”, “response to a therapy”, or “response to administration of a modulator” refers to the result of any kind of treatment on an individual, and includes beneficial, neutral, and adverse effects.

The term “therapeutically effective amount” refers to an amount of a therapeutic agent, which when administered alone or together with one or more additional therapeutic agents, induces the desired response, such as decreasing signs and symptoms associated with disease. Often, the therapeutically effective amount provides the desired response without causing significant side effects to the administered subject.

The term “disease-associated nucleic acid” refers to a nucleic acid that has been found to be associated or correlated with the disease. This includes markers and haplotypes described herein, and/or markers and haplotypes in strong linkage disequilibrium therewith.

The term “modulator” refers to a compound that affects the signaling, activity or expression of polypeptides or nucleic acid sequences (also referred to as “modulates”), and includes both activators and inhibitors. A modulator that increases or upregulates the signaling, activity or expression of polypeptides or nucleic acid sequences is referred to as an “activator”. A modulator that inhibits, reduces, decreases or downregulates the signaling, activity or expression of polypeptides or nucleic acid sequences is referred to as an “inhibitor”. “Modulation” refers to the act of modulating as defined above and can be performed with a modulator. Unless specified otherwise, “modulate” or “modulation” refers to the act of modulating as defined above, and includes both increasing or upregulating the signaling, activity or expression of polypeptides or nucleic acid sequences, as well as inhibiting, reducing, decreasing or downregulating the signaling, activity or expression of polypeptides or nucleic acid sequences.

The term “antisense modulator” refers to a modulator that affects the signaling, activity or expression of at least one nucleic acid sequence through some form of complementary binding or hybridization to the nucleic acid molecule. Common forms of antisense modulators include ASOs as well as nucleic acids used in the RNAi mechanism for gene modulation, including, but not limited to, miRNA, siRNA, and short hairpin RNA (shRNA).

The term “amplification” or to “amplify” refers to increasing the number of copies of a sequence of nucleotides. An example of amplification is the “polymerase chain reaction”, in which a sample containing sequences of nucleotides is contacted with a pair of oligonucleotide primers. The primers hybridize with a nucleotide sequence, are extended under suitable conditions, and then are dissociated from the nucleotide sequence. This process is repeated to increase the number of copies of a sequence of nucleotides. Other methods can be used for amplification and are known to a person with ordinary skill in the art.

The term “complementary” refers to complementary nucleobase pairing between two nucleotide sequences on either two different nucleotide strands or two regions of the same nucleotide strand. It is known in the art that adenine bases form complementary pairing with thymine or with uracil bases through the formation of specific hydrogen bonds. Likewise, cytosine bases form complementary pairing with guanine bases through the formation of specific hydrogen bonds. Other descriptions of complementary pairing are detailed herein.

The term “composition” refers to a compound that comprises one or more molecules. The composition can contain oligonucleotides, polypeptides, small molecules, other types of molecules, or a combination thereof. A “pharmaceutical composition” refers to a composition that includes a modulator, or at least one molecule considered to be a pharmaceutical agent.

The term “isolated” refers to a purified, enriched or concentrated population of molecules. “Isolated” also refers to the act of enriching or concentrating a particular molecule, compound or complex such that its purity is increased.

The term “tissue” refers to an aggregate of cells that form a specific physiological function in an organism.

The term “delivery”, when used in the context of drugs, agents, or pharmaceutical compositions, refers to the administration of a drug, agent, or pharmaceutical composition to an assay mixture, a cell in culture, an animal, or a human subject or patient.

A “carrier”, when used in the context of drugs, agents, or pharmaceutical composition is one or more molecules that is used to aid the delivery of one or more other molecules.

ALS is part of a broader spectrum of disorders known as “motor neuron disease” (MND) that includes primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), pseudobulbar palsy, Mills' syndrome, monomelic amyotrophy, post-polio syndrome (PPS), madras motor neuron disease (MMND), progressive muscular atrophy (PMA), spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), and progressive bulbar palsy (PBP). In one embodiment, the term ALS refers to the broader category of MND, and includes but is not limited to, PLS, HSP, pseudobulbar palsy, Mills' syndrome, monomelic amyotrophy, PPS, MMND, PMA, SMA, SBMA, and PBP.

ALS can share common mechanisms with other “neurodegenerative diseases”, “neuromuscular diseases” and “TDP-43 proteinopathies”, including frontotemporal lobar degeneration (FTLD) and frontotemporal dementia (FTD), Alzheimer's disease, Parkinson's disease, and retinal degeneration diseases such as age-related macular degeneration (AMD). In one embodiment, the term ALS refers to the broader category of neurodegenerative and neuromuscular diseases. In another embodiment, the term ALS refers to the broader category of diseases involving TDP-43 proteinopathy.

“Proteinopathies” refer to a class of diseases that can result from, in part or in whole, abnormal protein function or protein aggregates, which are caused by structural or configurational abnormalities, modifications to the protein sequence (e.g., post-translational modifications) or localization, leading to aggregation of those proteins as a consequence. Such abnormal protein function or aggregates can interrupt or alter normal cellular, tissue, or organ function.

“TDP-43 proteinopathies” refers to diseases wherein a sub-population of patients can exhibit an increase in levels of TDP-43 aggregates in the cytoplasm. Examples of which include but are not limited to, ALS, Alzheimer's disease, argyrophilic grain disease, vascular dementia, frontotemporal dementia (FTD, FTD-TDP-43, and FTD-tau) and the greater group of frontotemporal lobar degeneration (FTLD), semantic dementia, dementia with Lewy bodies, polyglutamine diseases, Huntington's disease, spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, parkinsonism, Parkinson's disease (PD), Perry syndrome, ALS-parkinsonism dementia complex of Guam, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), pseudobulbar palsy, Mills' syndrome, monomelic amyotrophy, post-polio syndrome (PPS), madras motor neuron disease (MMND), progressive muscular atrophy (PMA), spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), retinal degeneration diseases such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), and glaucoma. TDP-43 proteinopathies are further described in Gendron and Josephs (Gendron and Josephs, Neuropathol. Appl. Neurobiol. 36:97-112 (2010)), Lagier-Tourenne et al. (Lagier-Tourenne et al., Hum. Mol. Gen. 19(1): R46-R64 (2010)), and Matsukawa et al. (Matsukawa et al., Journal of Biological Chemistry, 291(45): 23464-23476 (2016)), the disclosures of which, together with references cited therein, are incorporated herein in their entirety.

“Retinal degeneration diseases” refer to a class of diseases that can result from, in part or in whole, damage to photoreceptor cells of the retina, resulting in a continuous decline in vision. The term “retinal degeneration diseases” can include diseases such as, age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma or vision loss associated with photoreceptor degeneration.

“Oxidative stress” refers to a condition where there is an excess production of reactive oxygen species (ROS) relative to antioxidants. The term “oxidative stress” includes diseases such as attention deficit hyperactivity disorder (ADHD), autism, Asperger syndrome, atherosclerosis, cancer, depression, myocardial infarction, cardiovascular disease, chronic fatigue syndrome, diabetes, fragile X syndrome, neurodegenerative diseases such as ALS, Huntington's disease, Parkinson's disease, Alzheimer's disease and multiple sclerosis; ophthalmological diseases such as glaucoma, cataract formation and macular degeneration; as well as liver injury, osteoporosis, autoimmune diseases, inflammatory diseases, stroke and sickle cell disease.

“Anemia” refers to a condition where there are insufficient healthy red blood cells to transport oxygen to the body's tissues, resulting in symptoms such as fatigue, weakness, and dizziness. Anemia can be an acute or chronic condition. There are different potential causes of anemia, including iron deficiency, vitamin deficiency, inflammation, aplastic anemia, bone marrow disease, hemolytic anemia or sickle cell anemia.

“Ischemic diseases” refer to vascular diseases involving an interruption or reduction in the supply of arterial blood to a tissue, or organ, resulting in tissue or organ damage. The term “ischemic disease” includes cardiovascular diseases such as cardiac ischemia, myocardial ischemia, ischemic cardiomyopathy, coronary artery disease, or myocardial infarction. The term “ischemic disease” also includes ischemic colitis, mesenteric ischemia, brain ischemia, stroke, renal ischemia, limb ischemia, peripheral vascular disease or cutaneous ischemia.

“Inflammatory diseases” refer to diseases that are characterized by inflammation. The term “inflammatory disease” includes but is not limited to, allergy, asthma, atherosclerosis, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), psoriasis, renal fibrosis, reperfusion injury, rheumatoid arthritis, transplant rejection, tubular ischemia-reperfusion damage or vascular inflammation. The term “inflammatory disease” also refers to neuroinflammatory diseases that are characterized by neurological damage caused by immune responses, including but not limited to, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, or other TDP-43 proteinopathies.

“Cancer” refers to an abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasize. The term “cancer” or “cancers” include but are not limited to oral, salivary, laryngeal, esophangeal, head and neck, lung, gastric, gallbladder, pancreatic, urothelial, bladder, renal, cervical, ovarian, prostate, breast or colorectal cancer; as well as fibrofolliculomas, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas (slow-growing tumors of the central nervous system), pancreatic neuroendocrine tumors, pheochromocytomas (noncancerous tumors of the adrenal glands), endolymphatic sac tumors, kidney cysts, or lung cysts.

“Ameliorating” is the lessening of severity of a disease, as measured by at least one indicator of that disease. Indicators can be symptoms of that disease, or a marker associated with the disease and can be objectively or subjectively evaluated. In certain embodiments, to “ameliorate” can mean to slow, halt, or reverse the progression of a disease.

A “dose” is a specified unit of a pharmaceutical composition that is provided for administration. In some embodiments, dose can refer to a specified amount of a pharmaceutical composition that is administered over a period of time. The dose can refer to the total amount of the pharmaceutical composition administered, or the amount of pharmaceutical composition administered per unit of time.

Modulation of FNIP1 and/or FNIP2

In certain embodiments, provided herein are compositions, systems and methods for modulating the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal, or human subject. In certain embodiments, provided herein are compositions, systems and methods for decreasing or inhibiting the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal, or human subject, in order to treat, prevent, or ameliorate a disease, particularly neuromuscular or neurodegenerative diseases, such as for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), and other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. In other embodiments, also provided herein are compositions, systems, and methods for modulating, in particular increasing or upregulating, the expression or activity of FNIP1 and/or FNIP2 in a cell, animal or human subject. Such compositions, systems, and methods can be used to prevent, ameliorate, or treat diseases, particularly inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers described herein.

FNIP1 encodes for a protein named folliculin interacting protein 1, which is also represented herein as FNIP1. FNIP1 is also known as KIAA1961. FNIP2 encodes for a protein named folliculin interacting protein 2, which is also represented herein as FNIP2. FNIP2 is also known as FNIP1-like protein, FNIPL, MAPO1, O6-methylguanine-induced apoptosis 1 protein, or KIAA1450. The FNIP1 or FNIP2 protein or polypeptide, referenced herein, includes any polymorphs and/or isoforms of the FNIP1 or FNIP2 protein respectively, for example, different protein products obtained from the expression of different nucleic acids encoding FNIP1 or FNIP2, such as described in SEQ ID NOs: 1-15. FNIP1 and/or FNIP2, as used herein, also refers to FNIP1 and/or FNIP2 genes or transcripts, such as described in SEQ ID NOs: 1-15. Furthermore, FNIP1 and/or FNIP2, as used herein, also refers to FNIP1 and/or FNIP2 genes or transcripts harboring one or more mutations, or FNIP1 and/or FNIP2 proteins obtained from the expression of such mutant genes or transcripts.

FNIP1 and FNIP2 are paralogs that can each dimerize with their binding partner folliculin (also known as FLCN) to form a FNIP1-FLCN complex or FNIP2-FLCN complex respectively. FNIP1, FNIP2 and FLCN are DENN (differentially expressed in normal and neoplastic cells) proteins comprising an N-terminal longin domain and a C-terminal DENN domain. The FNIP1-FLCN and FNIP2-FLCN complexes are formed by the interaction between the longin domains and interaction between the DENN domains of the respective members of the complex (Lawrence et al., Science 366: 971-977 (2019); Shen et al., Cell 179: 1-11 (2019)). The FNIP1-FLCN and/or FNIP2-FLCN complexes play important roles in regulating several pathways such as the Rag-mediated nutrient sensing pathway, the VHL-HIF-VEGF pathway, the TGF-β pathway, the autophagy pathway, the cell cycle, and RhoA signaling (Hasumi et al., PNAS 112(13): E1624-E1631 (2015)).

FNIP1, FNIP2 and FLCN are expressed in most tissues, for example, the brain, the skin and its appendages, the lungs, and the kidney, etc. One function of the FNIP1-FLCN complex and/or FNIP2-FLCN complex is as a tumor suppressor. Loss-of-function of FNIP1 and/or FNIP2, as well as mutations in FNIP1 or FNIP2, have been associated with cancers, such as for example, fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas, pancreatic neuroendocrine tumors, pheochromocytomas, endolymphatic sac tumors, kidney cysts and lung cysts. Inactivation of both FNIP1 and FNIP2 in the kidneys of mice results in enlarged polycystic kidneys and/or kidney cancer, which is similar to the phenotypes observed with FLCN-deficient kidneys. Furthermore, expression of one allele of either FNIP1 or FNIP2 in FNIP1/FNIP2 knockout mice can rescue the phenotype, thus suggesting that FNIP1 and FNIP2 are functionally redundant. Hasumi et al. (Hasumi et al., PNAS 112(13): E1624-E1631 (2015)) describes the roles of FNIP1 and FNIP2 in normal cells and in disease such as cancers, the disclosures of which, along with its references, are incorporated herein in its entirety.

Similarly, loss-of-function mutations in FLCN have been linked to kidney, lung and skin tumors, pneumothorax, and Birt-Hogg-Dube (BHD) syndrome. Schmidt et al. (Schmidt et al., Gene 640: 28-42 (2018)) describes the structure of FLCN, its normal roles in the cell and associated pathways, as well as its roles in diseases such as BHD, fibrofolliculomas, lung cysts, spontaneous pneumothorax and kidney tumors, the disclosures of which, along with its references, are incorporated herein in its entirety. Previously, Bastola et al. reported that VHL is a positive regulator of FLCN mRNA levels either by transcriptional induction, or stabilization of FLCN mRNA by repression of miRNAs that inhibit FLCN mRNA, or a combination of both (Bastola et al., PLoS ONE 8(7): e70030 (2013)). Loss-of-function mutations in VHL have been linked to von Hippel-Lindau (VHL) disease, hemangioblastomas (slow-growing tumors of the central nervous system), kidney cysts, clear cell renal cell carcinoma, pancreatic neuroendocrine tumors, pheochromocytomas (noncancerous tumors of the adrenal glands) and endolymphatic sac tumors. Gossage et al. (Gossage et al., Nature Reviews Cancer 15: 55-64 (2015)) describes the structure of VHL, its normal roles in the cell and associated pathways, as well as its roles in VHL disease and cancers, such as hemangioblastomas, kidney cysts, clear cell renal cell carcinoma, pancreatic neuroendocrine tumors, pheochromocytomas and endolymphatic sac tumors, the disclosures of which, along with its references, are incorporated herein in its entirety. Therefore, provided herein are compositions, systems and methods for increasing the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal, or human subject, in order to rescue loss-of-function in the FNIP, FLCN or VHL pathways, thereby treating, preventing or ameliorating a disease, such as von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, as well as cancers such as fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas, pancreatic neuroendocrine tumors, pheochromocytomas, endolymphatic sac tumors, kidney cysts, lung cysts.

Furthermore, loss-of-function mutations in FNIP1 in mice leads to B cell deficiency and the development of cardiomyopathy, which is similar to the effects of upregulating AMPK in mice and humans (Siggs et al., PNAS 113(26): E3706-E3715 (2016)). In addition, loss of FNIP1 in mice is associated with increased expression of inflammatory markers (Centini et al., PLoS One 13(6): e0197973 (2018)). Therefore, provided herein are compositions, systems and methods for increasing the expression or activity of FNIP1 and/or FNIP2 in a cell, an animal, or human subject, in order to treat, prevent or ameliorate a disease, such as inflammatory diseases, B cell deficiency and cardiomyopathy.

Thus, in certain embodiments, provided herein are modulators and their methods of use to increase the expression or activity of FNIP1 and/or FNIP2. In one embodiment, the modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and increases its transcription. In one embodiment, the modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and stabilizes it. In one embodiment, the modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and increases its translation. In some embodiments, the upregulation of FNIP1 and/or FNIP2 expression or activity can lead to an increase in FLCN expression or activity. In one embodiment, the modulator targets FNIP1 and/or FNIP2 protein and promotes its interaction with the FLCN protein. In one embodiment, the modulator targets the longin domain of FNIP1 and/or FNIP2 protein and promotes its interaction with the longin domain of FLCN. In one embodiment, the modulator targets the DENN domain of FNIP1 and/or FNIP2 protein and promotes its interaction with the DENN domain of FLCN. In one embodiment, the modulator targets the FNIP1 and/or FNIP2 protein and increases its activity in positively regulating the expression or activity of FLCN. Thus, in some embodiments, the increase in FNIP1 and/or FNIP2 expression or activity can be used to treat, prevent or ameliorate diseases such as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, B cell deficiency, cardiomyopathy, spontaneous pneumothorax, as well as cancers such as fibrofolliculomas and kidney tumors. In certain embodiments, the modulators provided herein that can increase the expression or activity of FNIP1 and/or FNIP2 include, but are not limited to antisense modulators, nucleic acid vectors, oligonucleotide modulators, peptide modulators, antibody modulators, and small molecule modulators. In some embodiments, the modulators provided herein can be used as prophylaxis to prevent diseases such as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, B cell deficiency, cardiomyopathy, spontaneous pneumothorax, as well as cancers. In other embodiments, the modulators provided herein can be used as therapeutics to treat or ameliorate the symptoms of diseases such as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, B cell deficiency, cardiomyopathy, spontaneous pneumothorax, as well as cancers such as fibrofolliculomas and kidney tumors.

The present invention is, in part, related to the novel discovery that instead of upregulating FNIP1 and/or FNIP2, which can be effective to treat diseases such as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, B cell deficiency, cardiomyopathy, spontaneous pneumothorax, as well as cancers such as fibrofolliculomas and kidney tumors, counterintuitively, reducing or inhibiting the expression or activity of FNIP1 and/or FNIP2 can be used to treat a unique set of diseases, particularly neuromuscular or neurodegenerative diseases, such as for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AIMD), and other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease.

Previously, FLCN has been shown to interact with TDP-43 in human embryonic kidney (HEK293) cells and to mediate the shuttling of TDP-43 from the nucleus into the cytoplasm (Xia et al., Human Molecular Genetics, 25(1): 83-96 (2016)). Given that the experiments in Xia et al. were performed in kidney cells, and that cellular function and disease mechanisms are typically unique across different tissues or cell types, there remains a need for investigations into the relationship of FLCN to cytoplasmic TDP-43 accumulation in neuronal cell types. Experiments have been performed by the inventors in disease-relevant neuronal cell types (e.g. motor neuron cells), which have led to the novel discovery that inhibiting or reducing the expression or activity of FLCN in neuronal cell types, which are implicated in neuromuscular or neurodegenerative diseases such as ALS and other TDP-43 proteinopathies, lead to a decrease in cytoplasmic TDP-43 aggregates (see Example 5) and an increase in cell survival (see Example 4). Given that FNIP1 and/or FNIP2 positively regulates the expression or activities of FLCN, therefore, inhibiting or reducing the expression or activity of FNIP1 and/or FNIP2 can lead to a decrease in FLCN expression or activity, which can lead to a decrease in the shuttling of TDP-43 from the nucleus into the cytoplasm, thereby leading to a decrease in cytoplasmic TDP-43 aggregates and an increase in cell survival. Thus, in certain embodiments, reducing or inhibiting the expression or activity of FNIP1 and/or FNIP2 can be used to treat, prevent, or ameliorate neuromuscular or neurodegenerative diseases, such as for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AIMD), and other TDP-43 proteinopathies.

Further support for this discovery can also be found in studies showing that loss of FNIP1-FLCN or FNIP2-FLCN in BHD patients lead to the constitutive activation of AMPK, which results in PGC-1α mediated mitochondrial biogenesis, induction of HIF-1α transcriptional activity and increased transcription of VEGF (Preston et al., Oncogene 30: 1159-1173 (2011); Yan et al., J Clin Invest. 124(6): 2640-2650 (2014)). While increased expression or activity of VEGF and/or HIF-1α contributes to tumorigenesis, counterintuitively, these changes can be beneficial to treat, prevent, or ameliorate neuromuscular or neurodegenerative diseases, such as for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), and other TDP-43 proteinopathies. VEGF plays important roles in neurogenesis, neuronal survival, neuronal migration, and axon guidance. Strategies to increase the expression or activity of VEGF or HIF-1α have been used in exploratory clinical studies, and have shown promising results to treat ALS and other neurological disorders (Pronto-Laborinho et al., BioMedResearch International, Article ID 947513 (2014)).

In certain embodiments, reducing or inhibiting the expression or activity of FNIP1 and/or FNIP2 stimulates mitochondrial biogenesis and angiogenesis via the activation of the VEGF and HIF-1α pathways, which can be used to treat, prevent or ameliorate anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. In addition, FLCN-deficient mice exhibit reduced inflammation when subject to a NASH-inducing diet, while FLCN-deficient nematodes develop increased resistance to oxidative stress and pathogens (Paquette et al., bioRxiv DOI: 10.1101/2020.09.10.291617 (2020)). Thus, in certain embodiments, reducing or inhibiting the expression or activity of FNIP1 and/or FNIP2, which results in a decrease in expression or activity of FLCN, can be used to treat, prevent or ameliorate inflammatory diseases and oxidative stress. Furthermore, knockout of FLCN in mice adipocytes results in resistance to obesity induced by a high-fat diet (Paquette et al., bioRxiv DOI: 10.1101/2020.09.10.291617 (2020)). Thus, in certain embodiments, reducing or inhibiting the expression or activity of FNIP1 and/or FNIP2, which results in a decrease in expression or activity of FLCN, can be used to treat, prevent or ameliorate obesity.

Thus, in certain embodiments, provided herein are modulators and their methods of use to reduce or inhibit the expression or activity of FNIP1 and/or FNIP2. In one embodiment, the modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and inhibits its transcription. In one embodiment, the modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and degrades or destabilizes it. In one embodiment, the modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and inhibits its translation. In some embodiments, the inhibition of FNIP1 and/or FNIP2 expression or activity can lead to a decrease in FLCN expression or activity. In one embodiment, the modulator targets FNIP1 and/or FNIP2 protein and blocks its interaction with the FLCN protein. In one embodiment, the modulator targets the longin domain of FNIP1 and/or FNIP2 protein and blocks its interaction with the longin domain of FLCN. In one embodiment, the modulator targets the DENN domain of FNIP1 and/or FNIP2 protein and blocks its interaction with the DENN domain of FLCN. In one embodiment, the modulator targets the FNIP1 and/or FNIP2 protein and inhibits its activity in positively regulating the expression or activity of FLCN. In yet another embodiment, the modulator targets the FNIP1 and/or FNIP2 protein and degrades or destabilizes it, resulting in a decrease in expression or activity of FLCN. In some embodiments, the inhibition of FNIP1 and/or FNIP2 expression or activity can lead to either a decrease in the level of pathological TDP-43 aggregates in the cytoplasm, or an increase in levels of functional TDP-43 in the nucleus, or a combination thereof, in order to treat, prevent or ameliorate neurodegenerative or neuromuscular diseases, such as for example, ALS or other TDP-43 proteinopathies. In certain embodiments, provided herein are modulators that disrupt the interaction between the FNIP1-FLCN complex and/or the FNIP2-FLCN complex and TDP-43. In some embodiments, the modulators target the RRM2 domain of TDP-43, thereby blocking its interaction with the FNIP1-FLCN complex and/or FNIP2-FLCN complex. In other embodiments, the modulators target the region of the FNIP1-FLCN complex and/or FNIP2-FLCN complex that interacts with the RRM2 domain of TDP-43, thereby blocking its interaction with TDP-43. Such modulators that specifically disrupt the interaction between the FNIP1-FLCN complex and/or FNIP2-FLCN complex and TDP-43 can lead to beneficial outcomes such as either a decrease in the level of TDP-43 aggregates in the cytoplasm, or an increase in levels of functional TDP-43 in the nucleus, or a combination thereof, while allowing for the non-targeted domains in FNIP1, FNIP2, FLCN, or TDP-43, to perform their normal functions, thus reducing undesired side effects. In some embodiments, provided herein are modulators that inhibit the expression or activity of FNIP1 and/or FNIP2, which can lead to an increase in HIF-1α and/or VEGF levels and/or activity. In some embodiments, an increase in HIF-1α and/or VEGF levels can be used to treat, prevent or ameliorate neurodegenerative or neuromuscular diseases, such as ALS or other TDP-43 proteinopathies. In other embodiments, an increase in HIF-1α and/or VEGF levels can be used to treat, prevent or ameliorate anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. In certain embodiments, the modulators provided herein that can decrease the expression or activity of FNIP1 and/or FNIP2 include, but are not limited to antisense modulators, nucleic acid vectors, oligonucleotide modulators, peptide modulators, antibody modulators, and small molecule modulators. In some embodiments, the modulators provided herein can be used as prophylaxis to prevent neuromuscular or neurodegenerative diseases, such as for example, ALS, other TDP-43 proteinopathies, as well as oxidative stress, obesity, anemia or ischemic diseases. In other embodiments, the modulators provided herein can be used as therapeutics to treat or ameliorate the symptoms of neuromuscular or neurodegenerative diseases, such as for example, ALS, other TDP-43 proteinopathies, as well as oxidative stress, obesity, anemia or ischemic diseases. In a preferred embodiment, the modulators provided herein can be used as prophylaxis to prevent ALS. In another preferred embodiment, the modulators provided herein can be used as therapeutics to treat or ameliorate the symptoms of ALS.

Modulation of FNIP1 and/or FNIP2 Via Associated Genes or Pathways

In certain embodiments, the inhibition or downregulation of FNIP1 and/or FNIP2 mRNA or FNIP1 and/or FNIP2 protein can be achieved by targeting FNIP1 and/or FNIP2-associated genes or pathways. Such decrease in expression or activity of FNIP1 and/or FNIP2 can be useful to treat, prevent or ameliorate diseases, particularly neuromuscular or neurodegenerative diseases, such as for example, ALS, other TDP-43 proteinopathies, as well as oxidative stress, obesity, anemia or ischemic diseases. In certain embodiments, provided herein are modulators that reduce or inhibit the expression, activity or signaling of FNIP1 and/or FNIP2 by targeting and inhibiting at least one gene or pathway that positively regulates or increases the expression, activity or signaling of FNIP1 and/or FNIP2. In another embodiment, provided herein are modulators that reduce or inhibit the expression of FNIP1 and/or FNIP2 by targeting and increasing the expression or activity of at least one gene or pathway that negatively regulates or inhibits the expression of FNIP1 and/or FNIP2. In one embodiment, provided herein are modulators that reduce or inhibit the activity of FNIP1 and/or FNIP2, by targeting and inhibiting another gene or pathway that is responsible for positively regulating the activity of FNIP1 and/or FNIP2. In yet another embodiment, provided herein are modulators that reduce or inhibit the activity of FNIP1 and/or FNIP2 by increasing the expression or activity of at least one gene or pathway that is responsible for negatively regulating the activity of FNIP1 and/or FNIP2.

In certain embodiments, the increase in expression or activity of FNIP1 and/or FNIP2 mRNA or FNIP1 and/or FNIP2 protein can be achieved by targeting FNIP1 and/or FNIP2-associated genes or pathways. Such increase in expression or activity of FNIP1 and/or FNIP2 can be useful to treat, prevent or ameliorate diseases such as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers described herein. In certain embodiments, provided herein are modulators that increase the expression, activity or signaling of FNIP1 and/or FNIP2 by targeting and increasing the expression or activity of at least one gene or pathway that positively regulates or increases the expression, activity or signaling of FNIP1 and/or FNIP2. In another embodiment, provided herein are modulators that increase the expression of FNIP1 and/or FNIP2 by targeting and decreasing the expression or activity of at least one gene or pathway that negatively regulates or inhibits the expression of FNIP1 and/or FNIP2. In one embodiment, provided herein are modulators that increase the activity of FNIP1 and/or FNIP2, by targeting and increasing the expression or activity of another gene or pathway that is responsible for positively regulating the activity of FNIP1 and/or FNIP2. In yet another embodiment, provided herein are modulators that increase the activity of FNIP1 and/or FNIP2 by decreasing the expression or activity of at least one gene or pathway that is responsible for negatively regulating the activity of FNIP1 and/or FNIP2.

In yet other embodiments, provided herein are modulators that target FNIP1 and/or FNIP2 to affect the nucleocytoplasmic distribution of FLCN. In a preferred embodiment, provided herein are modulators that enhance the nuclear distribution of FLCN and reduce its localization to the cytoplasm. When FLCN is expressed on its own, it is mostly localized to the nucleus. However, when FNIP1 or FNIP2 is co-expressed with FLCN, FNIP1-FLCN or FNIP2-FLCN complexes are observed in the cytoplasm respectively. The regulation of FLCN nucleocytoplasmic shuttling by FNIP1 and FNIP2 is described in Takagi et al. (Takagi et al., Oncogene, 27: 5339-5347 (2008)), Baba et al. (Baba et al., PNAS, 103(42): 15552-15557 (2006)) and Hasumi et al. (Hasumi et al., Gene, 31: 415(1-2), 60-67 (2008)), the disclosures of which, along with their references, are incorporated herein in its entirety. In certain embodiments, provided herein are modulators that targets, removes or interferes with the longin or DENN domains of FNIP1 and/or FNIP2, in order to reduce or prevent the cytoplasmic shuttling of FLCN, thereby leading to either a decrease in pathological TDP-43 aggregates in the cytoplasm, or an increase in levels of functional TDP-43 in the nucleus, or a combination thereof. Furthermore, the activity of FNIP1 and/or FNIP2 in positively regulating the expression or activity of FLCN is regulated by post-translational modifications to FNIP1 and/or FNIP2. In particular, the phosphorylation of Ser938, Ser939, Ser941, Ser946, and/or Ser948 of FNIP1 and/or FNIP2 by casein kinase 2 (CK2) promotes its activity of positively regulating the expression or activity of FLCN. Conversely, protein phosphatase 5 (PP5) dephosphorylates FNIP1 and/or FNIP2, thereby promoting its ubiquitination and proteasomal degradation. Therefore, in some embodiments, provided herein are modulators that decrease the activity of CK2, and/or increase the activity of PP5, in order to decrease the expression or activity of FNIP1 and/or FNIP2. In other embodiments, provided herein are modulators that increase the activity of CK2, and/or decrease the activity of PP5, in order to increase the expression or activity of FNIP1 and/or FNIP2.

Antisense Modulators

In one embodiment, the modulator used to modulate the expression or activity of FNIP1 and/or FNIP2, is an antisense modulator. In one embodiment, the antisense modulator inhibits or decreases the expression or activity of a targeted nucleic acid or polypeptide, such as FNIP1 and/or FNIP2. In one embodiment, the antisense modulator is an antisense oligonucleotide (ASO). ASO in various embodiments can be in any format well known to a person skilled in the art. ASOs comprise an oligonucleotide sequence that is complementary to the coding sequence, otherwise known as the sense strand, of a targeted gene. When a targeted gene is transcribed into pre-mRNA or mRNA, the ASO binds to the mRNA, forming a double-stranded RNA molecule or an RNA/DNA complex. The double-stranded nature of the resulting RNA molecule or RNA/DNA complex prevents effective translation of the target mRNA, thereby reducing or preventing expression of the resulting polypeptide. Furthermore, double-stranded RNA or RNA/DNA complexes are subject to degradation and digestion by a collection of enzymes known as endonucleases, such as RNase H, thereby decreasing or inhibiting the expression of the resulting polypeptide. In certain embodiments, the ASO can effect a change in splicing patterns of the mRNA such that one exon is exchanged for another (i.e., splice-switching). If exons that are important for the expression or activity of the resulting polypeptide are swapped for exons that lack those functions, that will result in a decrease in expression or activity of the resulting polypeptide. In other embodiments, the ASO can effect a change in splicing patterns of the mRNA such that one or more exons, or portion thereof, is removed (i.e., exon-skipping). In one embodiment, exon skipping results in a shift in the reading frame during translation, leading to premature stop codons and a truncated protein that is degraded by nonsense-mediated decay (NMD), thus decreasing the expression of the resulting polypeptide. In another embodiment, the ASO can effect a change in splicing patterns of the mRNA such that one or more introns, or portion thereof, is retained (i.e., intron retention), which can lead to a decrease in protein expression or activity. In one embodiment, the ASO inhibits the expression of a targeted polypeptide or nucleotide sequence in part or in its entirety. In another embodiment, the ASO inhibits an enzyme that affects the function of a targeted polypeptide. In yet another embodiment, the ASO inhibits the activity of an RNA molecule. In another embodiment, the ASO modulator reduces the level of an RNA molecule, such as a noncoding RNA molecule, thereby affecting the expression of a targeted nucleic acid or polypeptide. In yet other embodiments, the ASO can modulate the stability and rate of degradation of the mRNA. Rinaldi & Wood (2018) (Rinaldi, C. and Wood M. J. A. Nature Review Genetics 14:19-21 (2018)) describe in more detail the functions and uses of ASO and chemical modifications for ASOs to promote effectiveness and stability in the therapeutic context. The Rinaldi & Wood (2018) reference, including all references cited therein, are incorporated herein in its entirety. In certain embodiments, methods and compositions for the delivery of antisense modulators into a cell, an animal, or a human subject described herein can be utilized to decrease the expression or activity of FNIP1 and/or FNIP2.

In other embodiments, antisense modulators described herein can increase or upregulate the expression or activity of nucleic acids and/or polypeptides of a targeted gene, such as FNIP1 and/or FNIP2. In one embodiment, those antisense modulators comprise antisense oligonucleotides (ASOs) and can include the characteristics, lengths, modifications, complexes, and conjugates described herein. In one embodiment, the antisense modulator targets and decreases the levels of a natural antisense transcript (NAT) that is responsible for downregulating a targeted gene, thereby increasing the expression of the targeted gene. In one embodiment, the antisense modulator targets and blocks a miRNA binding site present on the mRNA transcript of a targeted gene that is responsible for downregulating the targeted gene, thereby increasing the expression of the targeted gene. In one embodiment, the antisense modulator targets and decreases the levels of a miRNA that is responsible for downregulating the expression of a targeted gene, thereby increasing the expression of the targeted gene. In one embodiment, the antisense modulator targets a destabilizing motif present on the mRNA transcript of a targeted gene, thereby increasing the stability of the mRNA and leading to increased expression of the targeted gene. In one embodiment, the antisense modulator targets a polyadenylation signal motif on the mRNA transcript of a targeted gene, thereby increasing the stability of the mRNA and leading to increased expression of the targeted gene. In one embodiment, the antisense modulator targets an upstream open reading frame, thereby leading to increased expression of the targeted gene. In certain embodiments, methods and compositions for the delivery of antisense modulators into a cell, an animal, or a human subject described herein can be utilized to increase or upregulate the expression or activity of FNIP1 and/or FNIP2.

ASOs can be produced by any number of methods known to a person who is skilled in the art (see below for examples of some specific methods).

RNAi Modulators

In one embodiment, the modulator can be a therapeutic oligonucleotide used in the RNAi process. The therapeutic RNAi oligonucleotide can include miRNA, siRNA, or shRNA.

The oligonucleotides used in the RNAi process can be in any form well known to a person skilled in the art. The RNAi process involving siRNA/shRNA comprises several steps. First, a double-stranded oligonucleotide is introduced into the cell either exogenously or through the introduction of a viral vector that transcribes it. Second, the double-stranded oligonucleotide is cleaved by the ribonuclease protein Dicer into siRNA, which are short oligonucleotide fragments of around 20-25 base pairs. This step is not needed if synthetic siRNAs, which resemble the products of Dicer, are used. Third, the siRNA is separated by RISC into single-stranded oligonucleotide fragments, comprising the sense and antisense strands to a target RNA, and integrated into the RISC to form a RISC-siRNA complex. Fourth, the RISC-siRNA complex containing the antisense strand hybridizes to a target RNA that is complementary to it and cleaves the target RNA, thereby inhibiting translation of the target RNA into a polypeptide. In some embodiments, the oligonucleotides used in RNAi comprises preformed double-stranded (duplex) RNA, and which are preferably 19-29 nucleotides in length. In one embodiment, the oligonucleotide used in RNAi comprise a single-stranded, short hairpin RNA (shRNA), which consists of two complementary RNA sequences that are preferably 19-22 nucleotides each in length, and which are linked by a short loop of 4-11 nucleotides. The shRNA can be encoded in the form of DNA and delivered into cells via a nucleic acid vector, wherein it is transcribed to form the shRNA. miRNA are non-coding RNA sequences found endogenously that use the same RISC pathway to target and inhibit other RNA molecules. One difference between miRNA and siRNA is that miRNAs can operate by imperfect base pairing and typically affects multiple target RNAs, whereas siRNAs usually operate by perfect base pairing leading to specific knockdown of a target RNA. miRNAs are transcribed in the nucleus to give primary miRNA (pri-miRNA), which consists of a double-stranded stem-loop structure. Pri-miRNA is cleaved by a microprocessor complex to form precursor miRNA (pre-miRNA), which is 70-100 nucleotides in length. Pre-miRNA is transported from the nucleus to the cytoplasm, where it is processed by Dicer into a miRNA duplex of 18-25 nucleotides, which is incorporated into the RISC complex. The RISC-miRNA complex containing the antisense strand hybridizes to a target RNA that is complementary to it, resulting in translational repression, degradation and/or cleavage of the target RNA. RNAi processes and their therapeutic applications are described in more detail by Aagaard and Rossi (Aagaard L and Rossi JJ, Advanced Drug Delivery Reviews 59:75-86 (2007)), and Lam et al. (Lam et al., Molecular Therapy, Nucleic Acids 4, e252 (2015)), the disclosures of which, along with their references, are incorporated herein in their entirety.

In some embodiments, the RNAi oligonucleotide modulator inhibits or decreases the expression or activity of a targeted nucleic acid or polypeptide, such as FNIP1 and/or FNIP2. In other embodiments, the RNAi oligonucleotide modulator increases the expression or activity of a targeted nucleic acid or polypeptide, such as FNIP1 and/or FNIP2. In one embodiment, the RNAi oligonucleotide modulator inhibits or decreases the expression of a targeted polypeptide in part or in its entirety. In another embodiment, the RNAi oligonucleotide modulator inhibits a regulator that affects the expression or activity of a targeted polypeptide or nucleotide sequence. In another embodiment, the RNAi oligonucleotide modulator inhibits another nucleic acid, such as a noncoding RNA, which affects the expression or activity of the target polypeptide or nucleic acid sequence.

RNAi oligonucleotides can be produced by any number of methods known to a person who is skilled in the art (see below for specific methods).

Hybridization

In certain embodiments, the antisense modulators disclosed herein can hybridize with a target nucleic acid encoding FNIP1 and/or FNIP2. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleobases of the antisense modulator and target nucleic acid, such as, for example, Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. The conditions under which an antisense modulator can hybridize with a target nucleic acid molecule can vary. Under stringent conditions, an antisense modulator hybridizes in a sequence-dependent manner determined by the nature and composition of the nucleic acid molecules to be hybridized. Methods of determining whether an antisense modulator sequence can hybridize specifically to a target nucleic acid are well known in the art.

Target Nucleic Acids, Target Regions, Target Segments and Nucleobase Sequences

In certain embodiments, an antisense modulator comprises an oligonucleotide, or portions thereof, that can hybridize to a target nucleic acid, wherein the target nucleic acid encodes FNIP1 and/or FNIP2.

In certain embodiments, target nucleic acids can comprise nucleobase sequences encoding FNIP1 and/or FNIP2, including but not limited to the following: the reverse complement of RefSeq Accession No. NC_000004.12 truncated from nucleotides 158767000 to 158910000 (incorporated herein as SEQ ID NO: 1), RefSeq Accession No. NM_020840.3 (incorporated herein as SEQ ID NO: 2), RefSeq Accession No. NM_001323916.2 (incorporated herein as SEQ ID NO: 3), RefSeq Accession No. NM_001346043.2 (incorporated herein as SEQ ID NO: 4), RefSeq Accession No. NM_001366843.1 (incorporated herein as SEQ ID NO: 5), RefSeq Accession No. XM_017008487.1 (incorporated herein as SEQ ID NO: 6), RefSeq Accession No. XM_005263158.2 (incorporated herein as SEQ ID NO: 7), RefSeq Accession No. XM_005263160.3 (incorporated herein as SEQ ID NO: 8), RefSeq Accession No. XR_001741297.1 (incorporated herein as SEQ ID NO: 9), RefSeq Accession No. XM_024454161.1 (incorporated herein as SEQ ID NO: 10), RefSeq Accession No. NC_000005.10 truncated from nucleotides 131639000 to 131799000 (incorporated herein as SEQ ID NO: 11), RefSeq Accession No. NM_133372.3 (incorporated herein as SEQ ID NO: 12), RefSeq Accession No. NM_001008738.3 (incorporated herein as SEQ ID NO: 13), RefSeq Accession No. NM_001346113.2 (incorporated herein as SEQ ID NO: 14), and RefSeq Accession No. NM_001346114.2 (incorporated herein as SEQ ID NO: 15). In some embodiments, antisense modulators can also target other nucleobase sequences encoding FNIP1 and/or FNIP2 (e.g. other DNA sequences, cDNA sequences, scaffolds, or mRNA transcript variants), which can be found by accession number in databases such as NCBI and GENBANK, and which are incorporated herein by reference. In other embodiments, nucleobase sequences encoding FNIP1 and/or FNIP2 include previous and future versions of nucleobase sequences encoding FNIP1 and/or FNIP2, which can be found by accession number in databases such as NCBI and GENBANK, and which are also incorporated herein by reference. In other embodiments, nucleobase sequences encoding FNIP1 and/or FNIP2 include mRNA transcripts that are formed from the utilization of alternative polyadenylation sites.

The nucleobase sequence set forth in each SEQ ID NO contained herein is independent of any modification to a nucleobase, a sugar moiety, or an internucleoside linkage. In some embodiments, antisense modulators, or portions thereof, that are defined by a percent complementarity or percent identity to a nucleobase sequence set forth in a SEQ ID NO or sample reference number (GiTx ID #) described herein can comprise, independently, one or more modifications to a nucleobase, one or more modifications to a sugar moiety, or one of more modifications to an internucleoside linkage.

In certain embodiments, an antisense modulator can hybridize to at least one target region within the target nucleic acid. A target region is a structurally defined region of the target nucleic acid. Examples of a target region include but are not limited to an exon, an intron, an exon-intron junction, an intron-exon junction, an exon-exon junction, a 3′ untranslated region (3′ UTR), a 5′ untranslated region (5′ UTR), a translation initiation region, a translation termination region, a 5′ donor splice site, a 3′ acceptor splice site, a start codon, an upstream open reading frame (ORF), a repeat region, a hexanucleotide repeat expansion, a splice enhancer region, an exonic splicing enhancer (ESE), a splice suppressor region, an exonic splicing silencer (ESS), an intronic splicing enhancer (ISE), an intronic splicing silencer (ISS), a RNA destabilization motif, a miRNA binding site, or other defined nucleic acid region. The structurally defined regions for FNIP1 and/or FNIP2 can be obtained by accession number from sequence databases such as NCBI and GENBANK, and such information is incorporated herein by reference.

In some embodiments, a target region can contain one or more target segments. In some embodiments, multiple target segments within a target region can be non-overlapping. In certain embodiments, target segments within a target region are separated by less than 5000, 2500, 1000, 500, 250, 100, 50, 40, 30, 20, 10, or 5 nucleotides. In other embodiments, target segments within a target region are overlapping or contiguous. In certain embodiments, an antisense modulator can hybridize to a 5′ target segment within a target region and a 3′ target segment within the same target region. In other embodiments, an antisense modulator can hybridize to a 5′ target segment within a target region and a 3′ target segment within a different target region.

A suitable target segment can specifically exclude a certain structurally defined target region, such as, for example, a start codon or a stop codon. The determination of suitable target segments can include a comparison of the nucleobase sequence of the target segment to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity amongst different sequences. This comparison can enable the selection of antisense modulator sequences that have increased specificity for a target segment and a corresponding reduced likelihood of hybridizing in a non-specific manner to non-target or off-target sequences.

In some embodiments, targeting includes determination of at least one target segment within a target nucleic acid to which an antisense modulator, or portion thereof, can hybridize in order to produce a desired effect. The desired effect can be a decrease or increase in mRNA levels of a target nucleic acid. The desired effect can also be a decrease or increase in levels of a protein encoded by the target nucleic acid. The desired effect can also be a phenotypic change associated with a change in mRNA levels of a target nucleic acid or change in protein levels encoded by the target nucleic acid. In certain embodiments, the desired effect of using an antisense modulator to target at least one target segment within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it hybridizes, is a reduction in FNIP1 and/or FNIP2 mRNA levels. In other embodiments, the desired effect of using an antisense modulator to target at least one target segment within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it hybridizes is a reduction in FNIP1 and/or FNIP2 protein levels. In yet other embodiments, the desired effect of using an antisense modulator to target at least one target segment within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it hybridizes is a phenotypic change associated with the reduction of FNIP1 and/or FNIP2 mRNA or protein levels. In certain embodiments, the desired effect of using an antisense modulator to target at least one target segment within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it hybridizes, is an increase in FNIP1 and/or FNIP2 mRNA levels. In other embodiments, the desired effect of using an antisense modulator to target at least one target segment within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it hybridizes is an increase in FNIP1 and/or FNIP2 protein levels. In yet other embodiments, the desired effect of using an antisense modulator to target at least one target segment within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it hybridizes is a phenotypic change associated with the increase of FNIP1 and/or FNIP2 mRNA or protein levels.

Targeting FNIP1 and/or FNIP2 Nucleic Acids

In certain embodiments, the antisense modulators described herein, or portion thereof, can hybridize to any target nucleic acid comprising nucleotide sequences encoding FNIP1 and/or FNIP2. In some embodiments, the antisense modulators can hybridize to target nucleic acids at any stage of RNA processing within the cell, for example, pre-mRNA or mature mRNA. In yet other embodiments, antisense modulators can hybridize to any target region(s) within the target nucleic acid, for example, an exon, an intron, a 5′ UTR, a 3′ UTR, a repeat region, a hexanucleotide repeat expansion, a miRNA binding site, a splice junction, an exon-exon junction, an exon-intron junction, an intron-exon junction, an exonic splicing silencer (ESS), an exonic splicing enhancer (ESE), an intronic splicing enhancer (ISE), an intronic splicing silencer (ISS), exon 1, exon 2, exon 3, intron 1, or intron 2, etc. In one embodiment, antisense modulators can hybridize to at least one exon present in SEQ ID NOs: 1-15. In other embodiments, antisense modulators can hybridize to target regions other than exons that are present in SEQ ID NOs: 1-15, wherein such regions are described in databases such as NCBI and GENBANK, which are incorporated herein by reference.

In certain embodiments, the antisense modulators described herein can hybridize to all RNA transcript variants of FNIP1 and/or FNIP2. In other embodiments, the antisense modulators described herein hybridize selectively to at least one RNA transcript variant of FNIP1 and/or FNIP2. Transcript variants of FNIP1 and/or FNIP2 can include mRNA transcripts generated by differential splicing, or mRNA transcripts containing mutations (e.g., SNPs, INDELs etc.) when compared to a reference sequence. In certain embodiments, the antisense modulators described herein inhibit the expression of all transcript variants of FNIP1 and/or FNIP2. In certain embodiments, the antisense modulators described herein inhibit expression of all transcript variants of FNIP1 and/or FNIP2 equally. In certain embodiments, the antisense modulator described herein preferentially inhibits the expression of certain transcript variants of FNIP1 and/or FNIP2. In other embodiments, the antisense modulators described herein increase the expression of all transcript variants of FNIP1 and/or FNIP2. In certain embodiments, the antisense modulators described herein increase the expression of all transcript variants of FNIP1 and/or FNIP2 equally. In certain embodiments, the antisense modulator described herein preferentially increases the expression of certain transcript variants of FNIP1 and/or FNIP2. In certain embodiments, antisense modulators described herein are useful for reducing cytoplasmic TDP-43 aggregates, or increasing the levels of functional TDP-43 in the nucleus, or a combination thereof. In certain embodiments, antisense modulators described herein are useful for normalizing the expression of various mis-regulated genes.

In certain embodiments, provided herein are antisense modulator sequences designed to target various regions of FNIP1 and/or FNIP2 transcripts produced by the FNIP1 gene (RefSeq Accession No. NC_000005.10 truncated from nucleotides 131639000 to 131799000, incorporated herein as SEQ ID NO: 11) and/or the FNIP2 gene (the reverse complement of RefSeq Accession No. NC_000004.12 truncated from nucleotides 158767000 to 158910000, incorporated herein as SEQ ID NO: 1). In some embodiments, the nucleotide sequence, target start site, target stop site, and description of example antisense oligonucleotide modulator sequences targeting FNIP1 are specified in Table 1. In other embodiments, the nucleotide sequence, target start site, target stop site, and description of example antisense oligonucleotide modulator sequences targeting FNIP2 are specified in Table 4. The predicted binding energy of each antisense modulator to the target sequence, as calculated using software known in the art, such as RNAstructure, are also described under “Binding Score” in Table 1 and Table 4 (see SEQ ID NOs: 16-1494). Antisense modulators with greater binding energy (more negative binding score) are predicted to hybridize better to the target sequence.

Complementarity

An antisense modulator is said to be complementary to a target nucleic acid, for example a target nucleic acid encoding FNIP1 and/or FNIP2, when one or more nucleobases of the antisense modulator can hydrogen bond with the corresponding complementary nucleobases of the target nucleic acid, such that the antisense modulator can hybridize in a sequence-dependent manner to the target nucleic acid. In certain embodiments, an antisense modulator can comprise one or more non-complementary nucleobases to the target nucleic acid, provided that the antisense modulator retains its ability to hybridize to the target nucleic acid. In certain embodiments, when two or more non-complementary nucleobases are present, they can be contiguous (i.e., linked) or clustered together. In other embodiments, when one or more non-complementary nucleobases are present, they can be interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. In certain embodiments, one or more non-complementary nucleobases can be located either at the 5′ end, 3′ end, internal region, or a mix of any regions of the antisense modulator. In one embodiment, a non-complementary nucleobase is present in the wing region of an antisense oligonucleotide modulator comprising a gapped sequence, as described in more detail below. An antisense modulator can also hybridize to one or more target segments in the target nucleic acid, such that adjacent or intervening segments do not take part in the hybridization event (e.g., forming a hairpin or loop structure, or mismatch).

In certain embodiments, provided herein are antisense modulators, or specified portions thereof, that are at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an equal length portion of a target nucleic acid, a target region, or a target segment encoding or associated with FNIP1 and/or FNIP2, such as described in SEQ ID NOs: 1-15. By way of example, an antisense modulator in which 16 of its 20 nucleobases are complementary to a target nucleic acid encoding FNIP1 and/or FNIP2, and would therefore specifically hybridize to it, would represent 80% complementarity. Methods to determine percent complementarity, percent homology, or sequence identity of an antisense modulator to a target nucleic acid, a target region, or a target segment, are well known in the art, for example, using basic local alignment search tools (BLAST) and PowerBLAST programs (Altschul et al., Journal of Molecular Biology, 215: 403-410 (1990); Zhang and Madden, Genome Research, 7: 649-656 (1997)), and the Gap program (Wisconsin Sequence Analysis Package) that relies on the Smith and Waterman algorithm (Smith and Waterman, Advances in Applied Mathematics, 2: 482 489 (1981)). These methods, along with their respective publications and those references cited within, are incorporated herein in their entirety.

In certain embodiments, provided herein are antisense modulators, or specified portions thereof, that are 100% complementary (i.e., fully complementary) to a target nucleic acid, a target region or a target segment, for example, encoding FNIP1 and/or FNIP2. As used herein, “fully complementary” means each nucleobase of an antisense modulator is capable of specific base pairing with complementary nucleobases of a target nucleic acid. By way of example, an antisense modulator that is 18 nucleobases long is said to be fully complementary to a target nucleic acid that is 3667 nucleobases long if all 18 nucleobases of the antisense modulator is capable of specific base pairing with complementary nucleobases of the target nucleic acid.

In some embodiments, complementarity can be determined for a specified portion of an antisense modulator. By way of example, a 20 nucleobase portion of a 35 nucleobase antisense modulator can be said to be fully complementary to a target nucleic acid that is 3667 nucleobases long if the 20 nucleobase portion can undergo specific base pairing with complementary nucleobases of the target nucleic acid. At the same time, in some embodiments, the entire 35 nucleobase antisense modulator can be fully complementary to the target nucleic acid sequence if the remaining 15 nucleobases of the antisense modulator are also complementary to the target sequence. In other embodiments, the 35 nucleobase antisense modulator is not fully complementary to the target nucleic acid sequence if the remaining 15 nucleobases of the antisense modulator are not fully complementary to the target sequence.

In certain embodiments, antisense modulators that are up to 10, 15, 20, 25, 30, or 35 nucleobases in length comprise no more than 1, no more than 5, no more than 10, no more than 15, no more than 20, or no more than 25 non-complementary nucleobase(s) respectively, relative to a target nucleic acid, such as a target nucleic acid encoding FNIP1 and/or FNIP2, or a specified portion thereof.

In certain embodiments, provided herein are antisense modulators that are complementary to a specified portion of a target nucleic acid, as defined by a number of contiguous (i.e. linked) nucleobases within a target region or target segment of a target nucleic acid. In some embodiments, the antisense modulator is complementary to at least 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or more contiguous nucleobases of a target nucleic acid, for example encoding FNIP1 and/or FNIP2, or a range defined by any two of these values.

Identity

In certain embodiments, the antisense modulators provided herein can have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or oligonucleotide represented by a specific sample reference number (e.g., GiTx ID #), or portion thereof, disclosed herein. An antisense modulator is identical to a sequence disclosed herein if both possess the same nucleobase pairing ability to a complementary target nucleotide sequence, regardless of other modifications to the antisense modulator. For example, a DNA or other antisense modulator with nucleobase thymine at given position(s) is identical to an RNA sequence disclosed herein with nucleobase uracil at those equivalent position(s), since both thymine and uracil base pair with adenine. In another example, an RNA or other antisense modulator with nucleobase uracil at given position(s) is identical to a DNA sequence disclosed herein with nucleobase thymine at the equivalent positions(s). Other examples include the use of synthetic nucleobases that have the same nucleobase pairing ability as standard nucleobases, such as that of adenine, guanine, thymine, cytosine, and uracil. In certain embodiments, antisense modulators that are lengthened and shortened versions of the oligonucleotides and nucleotide sequences disclosed herein are contemplated. In certain embodiments, antisense modulators that have non-identical nucleobases relative to the oligonucleotides and nucleotide sequences disclosed herein are also contemplated. The non-identical nucleobases can be contiguous (i.e. linked or adjacent) with each other or dispersed throughout the antisense modulator. The percent identity of an antisense modulator relative to a sequence disclosed herein is derived by calculating the percentage of nucleobases of the antisense modulator that can undergo identical base pairing as compared with the sequence disclosed herein.

In certain embodiments, antisense modulators, or portions thereof, are at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an equal length portion of one or more particular nucleotide sequence described in SEQ ID NOs: 16-1494, or oligonucleotide represented by a GiTx ID #, or portion thereof disclosed herein. In certain embodiments, provided herein are antisense modulators that are identical to a specified portion of a nucleobase sequence, as defined by a number of contiguous (i.e. linked) nucleobases in the nucleobase sequence. In some embodiments, the antisense modulator consists of at least 8 consecutive nucleobases with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to any of the nucleobase sequences of SEQ ID NOs: 16-1494. In other embodiments, the antisense modulator consists of at least 12 consecutive nucleobases with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to any of the nucleobase sequences of SEQ ID NOs: 16-1494.

Modifications

Modifications to an antisense modulator can be made to increase its efficacy, stability, and/or ease of administration, as well as decrease toxicity or other harmful side effects. Certain embodiments of the invention can include such modifications, which include, but are not limited to, modifications of the backbone or internucleoside linkages, modifications of the sugar component, modifications of the nucleobase component, or other modifications of the structure or chemistry of the nucleotide. All modulators and their modifications mentioned herein include salts, mixed salts, esters, salts of esters, and free acid or base forms. Certain embodiments can include a combination of one or more of the modifications mentioned below. Certain embodiments can include a combination of one or more of the modifications mentioned below with one or more unmodified nucleotides. All modifications to nucleotides mentioned herein that remove or otherwise modify naturally occurring components of nucleotides are still considered nucleotides, respectively. By way of example, a nucleotide that is missing a phosphorus or phosphate component, or that has its natural phosphate internucleoside linkage replaced by a phosphorothioate linkage for instance, or that has a ribose or deoxyribose component replaced by a modified sugar component, is still considered a nucleotide. Likewise, an oligonucleotide comprising a chain of at least two nucleotides, including the modified nucleotides described herein, is still considered an oligonucleotide, even if the component nucleotides in the oligonucleotide are covalently bonded in a manner that is modified from that of the naturally found phosphodiester bond. By way of example, a compound formed by two modified nucleotides covalently bonded with a phosphorothioate bond, is considered an oligonucleotide.

Modifications to an antisense modulator can be made along the backbone (i.e., linkages between different nucleosides). In one embodiment, the modification is the inclusion of a modified phosphoester group. In one embodiment, the modification is the inclusion of a phosphorothioate group or linkage. Phosphorothioate linkages have been shown to increase the resistance of antisense modulators to nucleases and thus increase their overall stability, while also able to maintain cleavability by certain ribonucleases (RNases), such as RNase H, once paired with a complementary or near-complementary oligonucleotide strand, thus increasing effectiveness in certain antisense applications (Eckstein F, Nucleic Acid Therapeutics, 24(6), 374-387 (2014)). This resistance has been found in both DNA and RNA and can be implemented in a wide range of nucleotide-based therapeutic modalities, including both ASO and RNAi therapies. In addition, phosphorothioate linkages can increase bioavailability of the modulator in certain cases by improving overall cellular uptake of the modulator.

Other embodiments of backbone modifications include the use of other phosphorothioate-based linkages, such as chiral phosphorothioate linkages, phosphorodithioate linkages, and phophorotrithioate linkages; phosphotriesters and alkylphosphotriesters; phosphonates, such as chiral phosphonates, 3′- and 5′-alkylene phosphonates, methylphosphonates (including 5′-O-methylphosphonate and 3′-O-methylphosphonate), hydroxyphosphonates, thionoalkylphosphonates, and other alkyl phosphonates; phosphonoacetate and thiophosphonoacetate linkages; phosphoroamidates, such as N3′-P5′ phosphoramidates, cationic phosphoramidates, methoxyethyl phosphoramidates, aminoalkylphosphoramidates, thiophosphoramidates, dithiophosphoramidates, thionophosphoramidates, and methanesulfonyl phosphoramidates; phosphonoamidate and phosphonothioate linkages; other thio-linked backbones; other phosphate backbones, such as 3′-5′ linked and 2′-5′-linked selenophosphates and boranophosphates, as well as those phosphates with inverted polarity with 3′-3′, 5′-5′, or 2′-2′ linkages; phosphinate linkages; acetyl- and thioacetyl-based linkages; other sulfur-containing backbones, such as sulfonate, sulfamide, sulfamate, sulfone, sulfoxide, and sulfide; silicon-containing backbones, such as siloxane and silyl; other heteroatom backbones such as those backbones containing N, O, or S; linkages with mixed heteroatoms; CH₂-containing linkages; carbonate-modified linkages; carboxymethyl-modified linkages; carbamate-modified linkages; short chain alkyl or cycloalkyl linkages; alkene- and azide-containing linkages; formacetal thioformacetal, methylene formacetal, and methylene thioformacetal linkages; methyleneimino linkages; hydrazino- and methylhydrazino-based linkages; methylene methylimino linkages; 5′-5′ linkages exposing two 3′ ends, as described by Bhagat et al. (Bhagat L et al., Journal of Medicinal Chemistry, 54(8): 3027-3036 (2011)), which, along with its references, is incorporated herein in its entirety; and other backbones known to those skilled in the art. In one embodiment, the modulator includes one of the backbone modifications described herein or other backbone modifications as known by a person skilled in the art.

Modifications to an antisense modulator can be made by modifying the sugar component of the modulator molecule. This sugar component is referred to as ribose or deoxyribose of RNA and DNA, respectively, but can also include other sugar components in other nucleotides. In one embodiment the sugar component of at least one nucleotide of the modulator is modified to include a 2′-O-methoxyethyl group (also referred herein as 2′-MOE). The 2′-MOE modification has been demonstrated to enhance nuclease resistance, as well as to lower cell toxicity and increase binding affinity with the desired modulator target. In one embodiment, the 2′-MOE modification includes a 2′, 4′-constrained 2′-MOE modification, as described by Pallan et al. (Pallan P S et al., Chemical Communications, 48(66): 8195-8197 (2012)), which, along with its references, is incorporated herein in its entirety. In one embodiment, the 2′ OH-group of at least one nucleotide in the modulator is replaced by at least one of H, SH, F, Cl, Br, I, NH₂, or ON. In another embodiment the 2′ OH-group of at least one nucleotide in the modulator is replaced by at least one of R, SR, NHR, or NR₂. R is defined, in this case, as one of C₁-C₆ alkyl, alkenyl, or alkynyl. In another embodiment, the sugar modification of the modulator is a 2′-O-methyl (2′-OMe) modification. In one specific embodiment, a 2′-OMe or other 2′-O-alkyl group modification is made to sugar groups of the modulator located at the two nucleotides closest to the 5′ end of the sequence. In the case of an RNAi modulator, this modification can be made on the 5′ end of both the sense and anti-sense strands, as described by U.S. Pat. No. 7,834,171, which, along with its references, is incorporated herein in its entirety. In certain embodiments, the sugar component of at least one nucleotide of the modulator is modified to include a bicyclic sugar, such as a 4′-CH(R)—O-2′ or 4′ (CH₂)₂—O-2′, wherein R is independently selected from H, C1-C12 alkyl, or a protecting group. In one embodiment, the sugar component of at least one nucleotide of the modulator is modified to include a bicyclic sugar consisting of a 4′-CH(CH₃)—O-2′ bridge or constrained ethyl (cEt) modification. A cEt modification can improve the effectiveness and allele selectivity of the antisense modulator and is further described by Pallan et al. In another embodiment, the modification of at least one nucleotide includes a tetrahydropyran-modified nucleoside. Other embodiments include the modification of at least one nucleotide of the antisense molecule to include a 2′-dimethylaminooxyethoxy group or 2′-dimethylaminoethoxyethoxyl group or other moieties obvious to those skilled in the arts.

Modifications to an antisense modulator can be made by modifying the nucleobase component of the modulator molecule. The nucleobase component refers to the nitrogen-containing compounds that, along with the sugar component, form nucleosides. Nucleobases found in nucleotides and elsewhere include adenine, guanine, cytosine, thymine, uracil, and their isomers. Examples of other nucleobases or modifications to nucleobases can include 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, methylhypoxanthine, 1-methylcytosine, 2-O-methylcytosine, 2,6-diaminopurine, 6-methyl, 2-propyl, and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 2-F-adenine, 2-aminoadenine, 2-aminopyridine, 2-amino-6-hydroxyaminopurine, 2-deoxyuridine, 3-ethylcytosine, 3 methylcytosine, 6-hydroxyaminopurine, 6-hydroxymethyladenine, 2-pyridone, 5-halo, including 5-bromo, 5-trifluoromethyl, 5-chloro, 5-fluoro, and other 5-halo, uracils and cytosines, 5-propynyl uracil and cytosine and other alkynyl-modified pyrimidine bases, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 3-deazaguanine, 3-deazaadenine tricyclic pyrimidines, and O- and N-alkylation, including N6-methyladenosine and N6-carbamoylmethyladenine, 3,N4-ethenocytosine, 1N2-ethenoguanine, N2,3-ethenoguanine, N2, N2-dimethylguanosine, carboxylethylguanine, 4-methylcytosine, 5-carboxylcytosine, 5-formylcytosine, 5-hydroxymethylcytosine, 5-glucosylmethylcytosine, methylthymine, 5,6-dihydrothymine, 5-hydroxymethyluracil, formyluracil, carboxyluracil, tricyclic pyrimidines, phenothiazine cytidine, G-clamps, carbazole cytidine, and pyridoindole, cytidine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-amido-7-deazaguanine, 7-deazaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 8-azaguanine, 8-azaadenine, inosine, 8-oxoguanine. In one embodiment, at least one nucleobase in at least one nucleotide of the antisense modulator is modified or replaced with at least one of the nucleobases or modified nucleobases mentioned above. In one embodiment the modification of the antisense modulator is a substitution of at least one cytosine nucleobase with 5-methylcytosine. In one embodiment, all cytosine nucleobases of the antisense modulator are substituted with 5-methylcytosine. The 5-methylcytosine substitution can lower the immunogenicity of antisense modulators and is a modification that is preferred in many forms of antisense compounds. In another embodiment, the antisense modulator is modified to have both at least one 5-methylcytosine substitution and at least one 2′-MOE sugar modification.

In one embodiment, the antisense modulator is modified to comprise phosphorodiamidate morpholino oligonucleotides (also referred to herein as PMOs or morpholinos). A PMO can be constructed with at least one nucleoside comprising a sugar component, such as ribose or deoxyribose, that has been substituted with a methylenemorpholine ring. This modified nucleoside can be linked to others via a phosphorodiamidate linkage, in place of a phosphodiester linkage. PMOs have been demonstrated to be resistant to a variety of enzymes, including nucleases, esterases and proteases. PMOs, as uncharged molecules, also present limited interactions with charged molecules, such as proteins. These advantages can make PMOs a suitable modification for use in antisense applications, in which increased stability in vitro and in vivo and high target specificity can be important factors for consideration. Descriptions of morpholinos, as well as their properties and variations, included in some of the embodiments herein, can be found in U.S. Pat. Nos. 9,469,664 and 10,202,602, which, along with their references, are incorporated herein in their entirety.

In one embodiment, the antisense modulator is modified to comprise locked nucleic acids (LNA). LNAs are nucleotides that have a sugar component modified to comprise a 2′ O to 4′ C methylene linkage. These modified nucleotides provide higher resistance to cleavage by digestive enzymes such as nucleases, as well as present much higher binding affinities to complementary or near-complementary nucleic acid-based targets. In another embodiment, the antisense modulator is modified to comprise other bridged nucleic acids (BNA), such as the 2′, 4′-BNA^(NC)[N-Me] modification. Descriptions of LNAs, as well as their properties and variations, included in some of the embodiments herein can be found in U.S. Pat. No. 9,428,534, which, along with its references, is incorporated herein in its entirety.

In one embodiment, the antisense modulator is modified to comprise tricyclo-DNA (tcDNA). tcDNA modifications can provide several benefits, including improved nuclease resistance, binding stability and improved targeting. The structure and other pertinent information of tc-DNA included in some of the embodiments herein are described by Ittig et al. (Ittig, D et al., Artificial DNA PNA & XNA 1(1): 9-16 (2010)) and U.S. Pat. No. 10,465,191, which, along with their references, are incorporated herein in their entirety.

In one embodiment, the antisense modulator is a modified oligonucleotide containing a gap segment (also referred to herein as “gapped sequences”, and otherwise known as “gapmers”). These gapped sequences comprise a sequence of unmodified or modified nucleosides (also referred to herein as the “central sequence”) flanked on at least one end by at least one sequence of either unmodified or modified nucleosides (also referred to herein as the “wing sequence”). In one embodiment, the central sequence comprises unmodified DNA nucleosides, which when hybridized to a target RNA, allows for endonucleases such as RNase H to cleave the target RNA. In another embodiment, the central sequence comprises a mix of modified and unmodified nucleosides, which when hybridized to a target RNA, allows for RNase H cleavage. In one embodiment, the antisense modulator comprises a central sequence flanked by wing sequences at both the 5′ and 3′ ends of the central sequence, wherein at least one nucleoside of the wing sequences comprises a modified sugar. In one embodiment, the wing sequence is a combination of modified and unmodified nucleosides. In one embodiment, the wing sequences comprise nucleosides wherein each nucleoside comprises a modified sugar. In certain embodiments, the central sequence is chosen to consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 linked nucleosides. In certain embodiments, the wing sequences are each independently chosen to consist of 2, 3, 4, 5, or 6 linked nucleosides. In one embodiment, the central sequence consists of 10 linked nucleosides flanked by wing sequences at the 5′ and 3′ ends of the central sequence, wherein each wing sequence consists of 5 linked nucleosides. In one embodiment, the central sequence consists of 10 linked nucleosides flanked by wing sequences at the 5′ and 3′ ends of the central sequence, wherein each wing sequence consists of 4 linked nucleosides. In one embodiment, the central sequence consists of 10 linked nucleosides flanked by wing sequences at the 5′ and 3′ ends of the central sequence, wherein each wing sequence consists of 6 linked nucleosides. In one embodiment, the antisense oligonucleotide comprises a gapped sequence consisting of a central sequence of 10 deoxynucleotides, which are flanked on both sides by wing sequences each consisting of 4 2′-MOE modified nucleotides. In another embodiment, the antisense oligonucleotide comprises a gapped sequence consisting of a central sequence of 10 deoxynucleotides, which are flanked on both sides by wing sequences each consisting of 5 2′-MOE modified nucleotides. Gapped sequences can increase resistance to enzymes, such as nucleases, and, in some cases, reduce the need for phosphorothioate modifications. In one embodiment, the gapped sequence comprises a central sequence flanked by wing sequences containing at least one LNA or BNA modification. In another embodiment, the gapped sequence comprises a central sequence flanked by wing sequences comprising at least one nucleoside consisting of a 2′-MOE modified sugar. In one embodiment, the gapped sequence comprises a central sequence flanked by wing sequences comprising nucleosides wherein each nucleoside consists of a 2′-MOE modified sugar. In another embodiment, the gapped sequence comprises a central sequence flanked by wing sequences comprising at least one nucleoside consisting of tcDNA. In another embodiment, the gapped sequence comprises a central sequence flanked by wing sequences comprising at least one nucleoside consisting of a cEt modification. In one embodiment, the wing sequences can be one or more of a combination of the aforementioned modified sequences. Other gapped sequences included in the embodiments herein are described in U.S. Pat. Nos. 7,015,215 and 10,017,764, which are incorporated, along with their references, herein in their entirety.

Gapped sequences containing modified nucleosides in the central sequence can reduce cellular protein-binding and improve the therapeutic index of the antisense modulator, as described by Shen et al. (Shen et al., Nature Biotechnology, 37(6): 640-650 (2019)), which, along with its references, is incorporated herein in its entirety. In some embodiments, the central sequence of the gapped sequence comprises at least one nucleoside consisting of a modified sugar. In some embodiments, the second nucleoside of the central sequence from the 5′ end of the gapped sequence consists of a modified sugar, such as a cEt or 2′-OMe modified sugar. In another embodiment, the antisense oligonucleotide comprises a gapped sequence consisting of a central sequence of deoxynucleotides, which are flanked on both sides by wing sequences consisting of 2′-MOE modified nucleotides, wherein the second nucleotide of the central sequence from the 5′ end of the oligonucleotide contains a 2′-OMe sugar modification. In another embodiment, the antisense oligonucleotide comprises a gapped sequence consisting of a central sequence of 10 deoxynucleotides, which are flanked on both sides by wing sequences each consisting of 4 2′-MOE modified nucleotides, and wherein the second nucleotide of the central sequence from the 5′ end of the oligonucleotide contains a 2′-OMe sugar modification.

In one embodiment, the antisense modulator comprises a peptide nucleic acid (PNA). PNAs are modified nucleic acids that can be created through the substitution of the nucleotide backbone for a pseudopeptide backbone (e.g., N-(2-aminoethyl)-glycine), which links nucleosides together via peptide bonds. The lack of charged groups in the backbone of PNAs provide for higher affinity and specificity between the modified antisense modulator and its complementary or near-complementary oligonucleotide target. In one embodiment, the PNA comprises a GripNA compound (Active Motif, Inc., Carlsbad, Calif.). In another embodiment, the PNA is a phosphono-PNA molecule comprising an additional phosphate group, as described by Efimov et al. (Efimov, V A et al., Nucleic Acids Research, 26(2): 566-575 (1998)). In another embodiment, the PNA molecule contains charged groups to promote intracellular delivery (e.g., see Efimov, V A et al., Nucleosides, Nucleotides & Nucleic Acids, 24(10-12): 1853-1874 (2005). Certain types of PNAs included in the embodiments herein are described in U.S. Pat. No. 6,962,906 and Montazersaheb et al. (Montazersaheb S et al., Advanced Pharmaceutical Bulletin, 8(4): 551-563 (2018)), which are incorporated, along with their references, herein in their entirety.

In one embodiment, the antisense modulator includes at least one bifacial nucleotide, also known as a Janus base. A Janus base comprises two binding sites to a complementary nucleotide, which can be used to simultaneously bind to both sense and antisense strands of a target oligonucleotide through Watson-Crick bonding. Benefits of using a Janus base modification include potentially higher target specificity and higher levels of target deactivation or efficacy. Examples and descriptions of Janus bases can be found in Thadke et al. (Tadke SA, Communications Chemistry, 1(79) (2018)) and Asadi et al. (Asadi A, The Journal of Organic Chemistry, 72(2): 466-475 (2007)), both of which are incorporated herein, along with their references, in their entirety.

In one embodiment, the antisense modulator forms a chimeric compound. Chimeric compounds can comprise one of many configurations, including, but not limited, to RNA-DNA, PNA-DNA, PNA-RNA, and other modified or unmodified oligonucleotide analogues bound to other modified or unmodified oligonucleotides. Certain uses of chimeric compounds, for example, can include providing one region that confers improved nuclease resistance, while another region increases specificity of binding or binding stability to a complementary or near-complementary target. Other chimeric compounds can offer other combinations of benefits, including any of the benefits specified for the modifications mentioned above.

These modifications described herein and other modifications, including modifications to internucleoside backbones, sugars and nucleobases, as well as conjugates and methods of delivery described below, are described in part by Shen and Corey (Shen X and Corey DR, Nucleic Acids Research, 46(4): 1584-1600 (2018)), Smith and Rula (Smith CIE and Zain R, Annual Review of Pharmacology and Toxicology, 59: 605-630 (2019)), Khvorova and Watts (Khvorova A and Watts JK, Nature Biotechnology, 35(3): 238-248 (2017)), Manoharan (Manoharan M, Biochimica et Biophysica Acta, 1489(1): 117-130 (1999)), Chawla et al., (Chawla M et al., Nucleic Acids Research, 43(14): 6714-6729 (2015)), and U.S. Pat. Nos. 9,399,774 and 8,101,743, 7,407,943, all of which, along with each of their references, are incorporated herein in their entirety. Other modifications are known to those skilled in the art and are considered to be included in the embodiments herein.

Conjugates, Complexes, and Structures

Certain embodiments of the invention comprise antisense modulators conjugated or bonded to at least one other molecule, such as a peptide or polypeptide, lipid, sugar, nucleotide or oligonucleotide, other polymer, cleavage agent, transport agent, intercalating agent, molecular beacon, hybridization-triggered crosslinking agent, lipophilic agent, and hydrophilic agent. These conjugated or bonded complexes can provide a number of benefits to the antisense modulator, including, but not limited to, increased effectiveness or activity, improved delivery to specific tissues or cells, enhanced cellular uptake, lowered toxicity, resistance to nuclease degradation, increased half-life or residence time, enhanced pharmacodynamic or pharmacokinetic properties, and improved selective targeting of alleles, genes or other targets. Certain embodiments can include a combination of one or more of the complexes mentioned below.

In one embodiment, the antisense modulator is conjugated to a protein or other polyamide, amine, or similar molecule. In another embodiment, the antisense modulator is conjugated to a lipid, phospholipid, cholesterol or thiocholesterol, cholic acid, aliphatic chain, hexylamino-carbonyl-oxycholesterol, or other similar molecule. In another embodiment, the antisense modulator is conjugated to another organic molecule, such as an ether or thioether, steroid, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, adamantine acetic acid, palmityl, fluorescein, rhodamine, coumarin, dye or other marker molecule, or other polymer, such as polyethylene glycol. In another embodiment, the antisense modulator is conjugated to another drug or pharmaceutical agent used to treat, prevent or ameliorate the symptoms of ALS or other degenerative disease, such as edaravone, riluzole, dextromethorphan, quinidine sulfate, dexpramipexole, or baclofen in the case of ALS. In some embodiments, the antisense modulator is conjugated to another drug or pharmaceutical agent used to treat, prevent or ameliorate a TDP-43 proteinopathy. In some embodiments, the antisense modulator is conjugated to another drug or pharmaceutical agent used to treat, prevent or ameliorate cancer. In certain embodiments, the antisense modulator is conjugated to another drug or pharmaceutical agent used to treat, prevent or ameliorate oxidative stress, or obesity. In other embodiments, the antisense modulator is conjugated to another drug or pharmaceutical agent used to treat, prevent or ameliorate VHL disease, BHD syndrome, or spontaneous pneumothorax. In other embodiments, the antisense modulator is conjugated to another drug, such as for example, another drug alleviating pain or other symptoms or improving uptake or delivery, such as blood thinners (e.g., aspirin, warfarin), anti-inflammatory and pain relief drugs (e.g., COX inhibitors, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, pranoprofen, carprofen, indomethacin, folinic acid, taiprofenic acid, diclofenac, niflumic acid, diazepines or benzodiazepines, barbiturate); or antibacterial, antiviral, antibiotic, or other drug that promotes at least one benefit in a therapeutic setting, including treatment efficacy, symptom alleviation, drug tolerance, or side effect mediation. In another embodiment, the antisense modulator is conjugated to another agent promoting transport across cell membranes, such as those described in Letsinger et al. (Letsinger et al., PNAS, 86(17): 6553-6556 (1989)), Zhao et al. (Zhao et al., Current Opinion in Biomedical Engineering, 13: 76-83 (2020)) or another agent promoting transport across the blood-brain barrier, as described in PCT Patent WO89/10134. All three references, along with the references described therein, are incorporated herein in their entirety. In another embodiment, the antisense modulator is conjugated to one or more GalNAc residues, which are recognized by the asialoglycoprotein receptor resulting in efficient uptake into cells. In one embodiment, the antisense modulator is conjugated to a G-quadruplex, as described by PCT Patent WO2017188898, which, along with its references, is incorporated herein in its entirety. In another embodiment, the antisense modulator is conjugated to another compound with special electromagnetic or optical properties, such as a photo-labile protecting group, as described by PCT Patent WO2017157950, which, along with its references, is incorporated herein in its entirety. In yet another embodiment, the antisense modulator is conjugated on at least one terminus to at least one stabilizing group to enhance properties such as, for example, nuclease stability. In one embodiment, the stabilizing groups are cap structures such as, for example, inverted deoxy abasic caps. Other stabilizing groups that can be used to cap one or both ends of an antisense modulator to impart nuclease stability are described in PCT Patent WO2003004602, which, along with its references, is incorporated herein in its entirety. Other conjugates, included in the embodiments herein, are described by Benizri et al. (Benizri et al., Bioconjugate Chemistry, 30: 366-383 (2019)), which, along with its references, is incorporated herein in its entirety.

Methods of Delivery

In certain embodiments, provided herein are methods and compositions for the delivery of antisense modulators into a cell, an animal, or human subject. In one embodiment, the antisense modulators are capable of decreasing or inhibiting the expression or activity of FNIP1 and/or FNIP2. In another embodiment, the antisense modulators are capable of increasing the expression or activity of FNIP1 and/or FNIP2. Many of these methods and compositions are known to those skilled in the art. In other embodiments, the methods and compositions for the delivery of antisense modulators described herein are also applied, with suitable modifications in some cases, to the delivery of other types of modulators, such as for example, other oligonucleotide modulators, antibody modulators, peptide modulators, or small molecule modulators.

In one embodiment, the method of delivery of antisense modulators includes direct introduction, or transfection, into a cell, an animal, or a human subject, via a transfection reagent, such as a liposomal-based or amine-based transfection reagent. This method of delivery can be used with or without aforementioned modifications or conjugations to the antisense modulator. Certain modifications and conjugations can improve the rate of introduction or stability of the antisense modulator and are included in these embodiments. For example, implementing any of the aforementioned modifications that imbue the antisense modulator with increased nuclease resistance can increase stability of the antisense modulator during and after introduction into a cell, an animal, or a human subject. Another example is the conjugation of the antisense modulator with a charged molecule or amphiphilic molecule to promote delivery across the cell membrane. In another embodiment, the method of delivery is electroporation or permeabilization. In another embodiment, the method of delivery includes the use of a liposome. In another embodiment, the method includes other forms of lipid-mediated transport. In another embodiment, the method of delivery involves the use of membrane fusion. In another embodiment, the method of delivery includes the use of colloids containing polymeric particles or solutions of nanoparticles. Nanoparticles can include certain properties that assist in targeting certain areas for delivery or otherwise promote delivery, such as electromagnetic properties. In another embodiment, the method of delivery includes the use of chemical-mediated transport, including the use of calcium phosphate. In another embodiment, the method of delivery includes peptide-mediated transport, including the use of polylysine. In another embodiment, the method of delivery includes the use of endocytosis. In other embodiments, the method of delivery can include microinjections directly into cells. In one embodiment, antisense modulators are delivered naked without transfection reagents. Other methods and examples of methods are described by Dokka and Rojanasakul (Dokka and Rojanasakul, Advanced Drug Delivery Reviews, 44(1): 35-49 (2000)), Lochmann et al. (Lochmann et al., European Journal of Pharmaceutics and Biopharmaceutics, 58: 237-251 (2004)), Dong et al. (Dong et al., Advanced Drug Delivery Reviews, 144: 133-147 (2019)), and Juliano (Juliano, Nucleic Acids Research, 44(14): 6518-6548 (2016)), all of which are incorporated herein, along with each of their references, in their entirety.

In one embodiment, the method of delivery includes one or more of the common delivery methods used to deliver drugs, including, but not limited to, injections that are vascular, extravascular, into cerebral spinal fluid, into the blood or lymph, intrathecal, oral uptake, nasal delivery or otherwise as an inhalant, and transdermal uptake.

Nucleic Acid Vector

In certain embodiments, the method of delivery of antisense modulators described herein into a cell, an animal, or human subject, involves the use of nucleic acid vectors. Nucleic acid vectors in various embodiments are biological vehicles used for the transmission of genetic material from one location to another and can be in any format well known to a person skilled in the art. Genetic material can include, but is not limited to, DNA, RNA, mRNA, siRNA, miRNA, lncRNA, guide RNA (gRNA), or antisense oligonucleotides. These and other forms of genetic material are well known to a person of ordinary skill in the art, and are included in some embodiments herein. A nucleic acid vector can be in the form of a plasmid, cosmid, artificial chromosome, DNA or other cassette, phagemid, and the like. The genetic sequence contained within vectors can be created by DNA/RNA synthesis, and/or modified via DNA/RNA editing or splicing techniques that are known to a person who is skilled in the art. These vectors can be specific or nonspecific to a certain type of cell. In one embodiment, the nucleic acid vector is non-integrating, and otherwise known as an episomal vector (i.e., it does not integrate into the genome of the cell). In one embodiment, non-integrating vectors are useful for targeting post-mitotic cells that are no longer undergoing cell division, since as long as the episomal vector can be stably maintained, the modulator can be stably expressed. In another embodiment, the nucleic acid vector is an integrating vector. In one embodiment, integrating vectors are useful for targeting stem cells that are actively undergoing cell division, since genome integration ensures that the encoded modulator is not lost during cell division. A nucleic acid vector can be classified as a viral vector or non-viral vector, which allows for different methods of delivery into a target cell, both of which are included in some embodiments.

A viral vector comprises a sequence of nucleotides that can be delivered into a target cell via a genetically modified virus, such as a retrovirus, adenovirus, adeno-associated virus, lentivirus, pox virus, alphavirus, or herpes virus. In some embodiments, the virus can be encapsulated or attached to liposomes, polymersomes, dynamic polyconjugates, nanoparticle complexes and the like. Once introduced, the virus delivers the nucleic acid vector to the host cell as part of its natural replication cycle. The complete nucleic acid vector can be integrated into the genome of the host cell. In certain embodiments, the virus directly inserts particular nucleic acid sequences contained within the nucleic acid vector into the genome of the host cell. In other embodiments, non-integrating viral vectors can be used to introduce nucleic acids into a host cell that are not subsequently integrated into the genome.

Several methods can be used to facilitate the successful delivery of viral vectors in gene therapy, such as pseudotyping, adaptor targeting, genetic systems targeting, and others known in the art. In general, viral vectors need to be modified to facilitate successful transduction into target cells. In pseudotyping, a viral attachment protein that is compatible with the target cell but that is produced by a different virus is integrated into the viral vector. In adaptor targeting, a small molecule is developed that has strong binding affinity to both the vector and the target cell. In genetic systems targeting, the genetic incorporation of a protein or polypeptide into the vector facilitates the binding of the vector to the target cell. Viral vectors for gene therapy are further described by Waehler et al. (Waehler et al., Nature Reviews Genetics, 8:573-587 (2007)), which along with references cited therein, is incorporated by reference in its entirety and are known to a person of ordinary skill in the art.

Non-viral vectors can be delivered by any one of a number of non-viral delivery methods. Vectors modified with lipids such as phospholipid phosphatidylserine, DOTMA, DOSPA, DOTAP, DMRIE, cholesterol, DOPE, or any combination thereof, can be used to form liposomes to deliver genetic material. Vectors modified with polymers, such as poly(L-lysine), polyethylenimine, PEG, or any combination thereof, can also be used to deliver genetic material in polymersomes. In certain cases, the vector can be modified with a combination of lipid or polymer or other molecule that is known to a person of ordinary skill in the art. Other methods of delivery of nucleic acid vectors include but is not limited to the use of cell-penetrating peptides, physiologically compatible nanoparticles, dynamic polyconjugates, GalNAc, or stable nucleic acid-lipid particle formulations. In certain cases, unmodified nucleic acid vectors are taken up using natural processes for the uptake of nucleic acids by a cell, such as endocytosis. Non-viral vectors for gene therapy and certain methods of delivery are described further by Yin et al. (2014) (Yin et al., Nature Reviews Genetics, 15:541-555 (2014)), which along with reference cited therein, are incorporated by reference in its entirety and are known to a person of ordinary skill in the art.

Analysis of Antisense Modulator Activity

The antisense modulators disclosed herein can have variable activity, for example, as defined by percent reduction of target nucleic acid (e.g., RNA) levels, percent reduction of levels of proteins encoded by target nucleic acids, or percent reduction of the activity of proteins encoded by target nucleic acids. In certain embodiments, reductions in FNIP1 and/or FNIP2 RNA levels, which include, but are not limited to, RNA from the transcription of the FNIP1 and/or FNIP2 genes, and RNA used for FNIP1 and/or FNIP2 protein translation, are indicative of inhibition of FNIP1 and/or FNIP2 expression. In particular embodiments, reductions in levels of one or more FNIP1 and/or FNIP2 transcripts disclosed by SEQ ID NOs: 1-15 herein, is indicative of inhibition of FNIP1 and/or FNIP2 expression. In some embodiments, reductions in levels of FNIP1 and/or FNIP2 protein is indicative of inhibition of FNIP1 and/or FNIP2 expression. In particular embodiments, reductions in levels of one or more FNIP1 and/or FNIP2 proteins that are translation products of one or more FNIP1 and/or FNIP2 transcripts disclosed by SEQ ID NOs: 1-15 herein, is indicative of inhibition of FNIP1 and/or FNIP2 expression. In other embodiments, reductions in activity of FNIP1 and/or FNIP2 proteins that are translation products of one or more FNIP1 and/or FNIP2 transcripts disclosed by SEQ ID NOs: 1-15 herein is indicative of inhibition of FNIP1 and/or FNIP2 expression.

The antisense modulators disclosed herein can also have variable activity, for example, as defined by percent increase of target nucleic acid (e.g., RNA) levels, percent increase of levels of proteins encoded by target nucleic acids, or percent increase of the activity of proteins encoded by target nucleic acids. In certain embodiments, increases in FNIP1 and/or FNIP2 RNA levels, which include, but are not limited to, RNA from the transcription of the FNIP1 and/or FNIP2 genes, and RNA used for FNIP1 and/or FNIP2 protein translation, are indicative of an increase of FNIP1 and/or FNIP2 expression. In particular embodiments, increases in levels of one or more FNIP1 and/or FNIP2 transcripts disclosed by SEQ ID NOs: 1-15 herein, is indicative of an increase of FNIP1 and/or FNIP2 expression. In some embodiments, an increase in levels of FNIP1 and/or FNIP2 protein is indicative of an increase of FNIP1 and/or FNIP2 expression. In particular embodiments, an increase in levels of one or more FNIP1 and/or FNIP2 proteins that are translation products of one or more FNIP1 and/or FNIP2 transcripts disclosed by SEQ ID NOs: 1-15 herein, is indicative of an increase of FNIP1 and/or FNIP2 expression. In other embodiments, an increase in activity of FNIP1 and/or FNIP2 proteins that are translation products of one or more FNIP1 and/or FNIP2 transcripts disclosed by SEQ ID NOs: 1-15 herein is indicative of an increase of FNIP1 and/or FNIP2 expression.

Activity of FNIP1 and/or FNIP2 refers to one or more activities that are normally carried out by FNIP1 and/or FNIP2 transcripts or proteins, such as, for example, regulation of FLCN expression and/or activity, regulation of TDP-43, and regulation of various pathways such as the Rag-mediated nutrient sensing pathway, the VHL-HIF-VEGF pathway, the TGF-β pathway, the autophagy pathway, the cell cycle, and RhoA signaling etc. Activity of FNIP1 and/or FNIP2 also refers to one or more activities that are normally carried out by genes or pathways that are associated with FNIP1 and/or FNIP2, which includes for example, regulation of autophagy, shuttling of TDP-43 from the nucleus to the cytoplasm, regulation of cell-cell adhesion, regulation of nutrient sensing pathways, regulation of the mTOR pathway, regulation of the AMPK pathway, regulation of cell cycle, regulation of hypoxic response, regulation of angiogenesis, regulation of glycolysis, or regulation of other signaling or metabolic pathways.

In certain embodiments, the antisense modulators disclosed herein can selectively target and decrease the levels or activity of one or more particular FNIP1 and/or FNIP2 transcript variants and the proteins encoded by them, such decrease in levels or activity of one or more FNIP1 and/or FNIP2 transcript variants or proteins being indicative of a decrease of FNIP1 and/or FNIP2 expression or activity. In yet other embodiments, certain phenotypic changes produced as a result of administration of an antisense modulator to cells, animals, or human subjects, can be indicative of decrease of FNIP1 and/or FNIP2 expression or activity, for example, increased cell survival, increased levels of HIF-1α, or decreased levels of TDP-43 aggregates in the cytoplasm. In other embodiments, the antisense modulators disclosed herein can selectively target and increase the levels or activity of one or more particular FNIP1 and/or FNIP2 transcript variants and the proteins encoded by them, such increase in levels or activity of one or more FNIP1 and/or FNIP2 transcript variants or proteins being indicative of an increase in FNIP1 and/or FNIP2 expression or activity. In yet other embodiments, certain phenotypic changes produced as a result of administration of an antisense modulator to cells, animals, or human subjects, can be indicative of an increase in FNIP1 and/or FNIP2 expression or activity, for example, decrease in cell proliferation, decrease in angiogenesis, etc. In certain embodiments, the methods and compositions described herein for the analysis of the activity of an antisense modulator are applied directly or in modified form to the analysis of the activity of other types of modulators, such as, for example, other oligonucleotide modulators, antibody modulators, peptide modulators, and small molecule modulators.

In certain embodiments, the modulation of expression or activity of FNIP1 and/or FNIP2 transcripts or proteins can lead to changes in the expression or activity of other genes, mRNA, proteins, or pathways in the cell, wherein such changes are indicative of an increase or decrease of FNIP1 and/or FNIP2 expression or activity. FNIP1 and/or FNIP2 positively regulates the expression or activity of FLCN. Thus, in some embodiments, a decrease in FLCN expression or activity is indicative of inhibition of FNIP1 and/or FNIP2. Furthermore, changes in FLCN-regulated genes, mRNA, proteins, or pathways in the cell can be indicative of changes in FNIP1 and/or FNIP2 expression or activity. The FNIP1-FLCN and/or FNIP2-FLCN complex regulates the mTORC1 and AMPK pathways in a cell type-dependent manner, which is described by Khabibullin et al. (Khabibullin et al., Physiol Rep, 2(8): e12107 (2014)), which along with reference cited therein, are incorporated herein by reference in its entirety. Thus, in some embodiments, an increase in mTOR signaling or activation of the mTOR pathway in certain cell types, for example SAEC cells, is indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. In some embodiments, a decrease in mTOR signaling or inhibition of the mTOR pathway in certain cell types, for example HBE cells, is indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. In some embodiments, an increase in AMPK signaling or activity of the AMPK pathway in certain cell types is indicative of a decrease in FNIP1 and/or FNIP2 expression or activity, and vice versa. In other embodiments, a decrease in AMPK signaling or activity of the AMPK pathway in certain cell types is indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. FNIP1 and/or FNIP2 is a negative regulator of PPARGC1A/PGC1α and mitochondrial biogenesis, as described by Hasumi et al. (Hasumi et al., PNAS 112(13): E1624-E1631 (2015)). Thus, in some embodiments, an increase in expression or activity of PPARGC1A/PGC1α or an increase in mitochondrial biogenesis, are indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. The FNIP1-FLCN and/or FNIP2-FLCN complex inhibits the activity of TFEB and TFE3, which are transcription factors for autophagy genes, as described in Petit et al. (Petit et al., J Cell Biol., 202(7): 1107-22 (2013)), which along with reference cited therein, are incorporated herein by reference in its entirety. Thus, in some embodiments, an increase in activity of TFEB or TFE3 are indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. Furthermore, FLCN can inhibit the induction of autophagy by inhibiting the accumulation of LC3B, and promoting the accumulation of LC3C, as described by Bastola et al. (Bastola et al., PLoS ONE 8(7), e70030 (2013)), which along with reference cited therein, are incorporated herein by reference in its entirety. Thus, in some embodiments, an increase in levels of LC3B, or a decrease in levels of LC3C, or an increase in autophagy activity, can be indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. In some cases, FLCN can promote autophagy by interaction with GABARAP and ULK1, as described in Dunlop et al. (Dunlop et al., Autophagy, 10(10): 1749-1760 (2014)), which along with reference cited therein, are incorporated herein by reference in its entirety. Thus, in some embodiments, a decrease in autophagic flux can be indicative of a decrease of FNIP1 and/or FNIP2 expression or activity, and vice versa. Changes to other genes, mRNA, proteins, or pathways in the cell that result from inhibiting the expression or activity of FNIP1 and/or FNIP2 are well known to a person skilled in the art and are incorporated herein as indicative of inhibition of FNIP1 and/or FNIP2 expression or activity.

Analysis of RNA Levels

In certain embodiments, the inhibition of FNIP1 and/or FNIP2 expression by a modulator, such as an antisense modulator, can be assessed by measuring the decrease in levels of FNIP1 and/or FNIP2 RNA transcripts. In other embodiments, the increase of FNIP1 and/or FNIP2 expression by a modulator, such as an antisense modulator, can be assessed by measuring the increase in levels of FNIP1 and/or FNIP2 RNA transcripts. RNA analysis can be carried out on poly(A)+mRNA or total cellular RNA. Methods of RNA isolation are well known in the art and include, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols, or using an RNA extraction kit (Qiagen) etc. The target RNA levels can be quantified using methods well known in the art and include, for example, Northern blot analysis, competitive polymerase chain reaction (PCR), or reverse transcription followed by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions.

Prior to quantitative real-time PCR, the isolated RNA first undergoes a reverse transcription reaction to produce complementary DNA (cDNA), which is then used as the substrate for the real-time PCR amplification reaction. Reagents for reverse transcription and real-time PCR can be obtained commercially (e.g., Invitrogen, Carlsbad, Calif.). The reverse transcription reaction and real-time PCR reactions can be performed sequentially in the same sample well or in different sample wells. The levels of a target gene or RNA that are obtained by real-time PCR can be normalized using either total RNA levels quantified by, for example, RIBOGREEN (Invitrogen, Carlsbad, Calif.), or normalized using the expression level of a gene whose expression in the cell is more or less stable, such as cyclophilin A. Methods of RNA quantification using RIBOGREEN are described in Jones et al. (Jones et al., Analytical Biochemistry, 265: 368-374 (1998)), which together with the references cited therein, are incorporated herein in its entirety. A CYTOFLUOR 4000 instrument (PE Applied Biosystems) can be used to measure RIBOGREEN fluorescence. The expression levels of cyclophilin A can be quantified by real-time PCR within the same well as that used for quantifying the levels of target RNA (i.e., by performing a multiplex reaction) or by running it in a separate well.

Probes and primers that hybridize to a target nucleic acid encoding FNIP1 and/or FNIP2 can be designed using methods that are well known in the art, and can include the use of software, such as, for example, PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

In certain embodiments, the inhibition of FNIP1 and/or FNIP2 expression by a modulator, such as an antisense modulator, can be assessed by measuring the decrease in levels of FNIP1 and/or FNIP2 protein. In other embodiments, the increase of FNIP1 and/or FNIP2 expression by a modulator, such as an antisense modulator, can be assessed by measuring the increase in levels of FNIP1 and/or FNIP2 protein. Several methods for quantifying or measuring protein levels of FNIP1 and/or FNIP2 are well known in the art, such as Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunocytochemistry, fluorescence activated cell sorting (FACS), immunohistochemistry, protein activity assays, quantitative protein assays, bicinchoninic acid assay (BCA assay) also known as the Smith assay, and the like. Antibodies that are specific for a target protein, such as FNIP1 and/or FNIP2, can be generated using conventional monoclonal or polyclonal antibody generation methods well known in the art, or identified and obtained commercially from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.). Antibodies for the detection of mouse, rat, monkey, and human FNIP1 and/or FNIP2 are available commercially.

In Vitro Testing of Antisense Modulators

In certain embodiments, the antisense modulators described herein can be administered to cultured cells in vitro to evaluate their effects on the expression of target gene(s) or other phenotypes. In certain embodiments, the antisense modulators described herein can be administered to cultured cells in vitro to evaluate the effects of antisense modulators on FNIP1 and/or FNIP2 expression or activity. In certain embodiments, the antisense modulators provided herein can be administered to cultured cells in vitro to evaluate their effects on one or more phenotypes, such as, for example, cell survival, cell morphology, or levels of TDP-43 aggregates in the cytoplasm. In certain embodiments, other modulators described herein, such as, for example, other oligonucleotide modulators, antibody modulators, peptide modulators, or small molecule modulators can be administered to cultured cells in vitro to evaluate their effects on FNIP1 and/or FNIP2 expression or activity, or one or more phenotypes, such as, for example, cell survival, cell morphology, or levels of TDP-43 aggregates in the cytoplasm.

In some embodiments, the cultured cells can have an animal origin, for example, Sf9 insect cells, Chinese hamster ovary (CHO) cells, rat cell lines, mouse cell lines, or non-human primate cell lines etc. In other embodiments, the cultured cells can have a human origin, for example, human embryonic kidney-derived epithelial cells (HEK293 or HEK293T), HeLa cells, human neural cell lines, ReN-VM cells, human fibroblast cell lines, HepG2 cells, Hep3B cells, and primary hepatocytes etc. Examples of cultured cells include those that are described in the catalogs of commercial vendors, such as, for example, Clonetics Corporation, Walkersville, Md.; American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C. etc., and are incorporated by reference herein. Such cells are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Cells can be cultured and tested in multi-well plates, for example, 24-well, 48-well, 96-well, 384-well plates etc.

In certain embodiments, the cultured cells are human induced pluripotent stem cell (iPSC) lines or other human stem cell lines. In certain embodiments, the cultured cells are differentiated cells that are derived from iPSC or other stem cell lines using methods that are well known in the art. Such differentiated cells include but are not limited to motor neuron cells, upper motor neuron cells, lower motor neuron cells, astrocyte cells, glial cells, microglial cells, corticol neuron cells, endothelial cells, dopaminergic neuron cells, neural stem cells, oligodendrocyte cells, other brain cells, cardiomyocytes, other cardiac cells, skeletal muscle cells, vascular endothelial or smooth muscle cells, hepatocytes, other liver cells, pancreatic (3-cells, other kidney cells, lung cells etc. In some embodiments, the modulators, including antisense modulators, described herein are administered to more than one cell type that are cultured together (i.e., co-culture).

In one embodiment, the human iPSCs or other stem cells are derived from healthy individuals. In another embodiment, the human iPSCs or other stem cells are derived from individuals that are at risk of, or suspected to be afflicted with, or diagnosed with a neuromuscular or neurodegenerative disease, including but not limited to ALS, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), and other TDP-43 proteinopathies disclosed herein etc. In some embodiments, the human iPSCs or other stem cells are derived from individuals with familial ALS, including but not limited to individuals with known pathogenic mutations in ALS genes such as C9orf72, SOD1, STMN2, NEK1, TARDBP, FUS, VCP, OPTN, SQSTM1, UBQLN2, hnRNPA1, MATR3 etc. In some embodiments, the human iPSCs or other stem cells are derived from individuals with sporadic ALS, including those with and without known ALS-causing mutations. In yet other embodiments, human iPSCs or other stem cells can be modified in the lab to reproduce or mimic the diseased condition, for example, by the introduction of disease-causing mutations in known disease-relevant genes using genetic engineering techniques such as homologous recombination, CRISPR/Cas9, TALENs or zinc-finger nucleases, etc. Such iPSC or stem cell lines are termed isogenic disease cell lines. In the case of ALS, examples of isogenic cell lines include but are not limited to those containing known disease-causing mutations in ALS genes such as SOD1, TARDBP, and others (Hor et al., bioRxiv 713651 (2019)). In other embodiments, the human iPSCs or other stem cells are derived from individuals that are at risk of, or suspected to be afflicted with, or diagnosed with one or more diseases, including but not limited to oxidative stress, obesity, anemia, ischemic disease, inflammatory disease, VHL disease, BHD syndrome, spontaneous pneumothorax, or cancer.

The use of cultured cells of human origin to evaluate the effects of modulators, including antisense modulators, produces results with higher relevance to the human condition compared to the use of animal models, which often fail to recapitulate important aspects of the disease due to a lack of conservation of gene targets, pathways, physiology and systems between the animal model and humans (van Damme et al., Disease Models & Mechanisms, 10, 537-549 (2017)). Consequently, promising therapies that show a positive effect in preclinical studies using ALS animal models (e.g., SOD1 transgenic mice) have all failed to translate to success in human clinical trials (Mitsumoto et al., Lancet Neurology, 13: 1127-38 (2014)). Moreover, the use of human iPSCs or other stem cells derived from disease patients with different profiles and backgrounds (e.g., sex, age, ethnicity, disease sub-type, presence or absence of known ALS mutations, etc.) allows for the evaluation of the effects of modulators, including antisense modulators, described herein on a broad spectrum of patients.

Described herein and in the Examples are methods for the administration of antisense modulators to cultured cells, which can be modified appropriately for the administration of other modulators such as, for example, other oligonucleotide modulators, antibody modulators, peptide modulators, or small molecule modulators. In general, antisense modulators are administered to cultured cells when the cells are approximately 60-80% confluent. Transfection reagents that are commonly used to introduce antisense modulators into cultured cells are well known in the art and include, for example, LIPOFECTAMINE or LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense modulators are mixed with LIPOFECTAMINE or LIPOFECTIN in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense modulator and a LIPOFECTAMINE or LIPOFECTIN concentration that typically ranges from 2-12 μg/mL per 100 nM of antisense modulator. Another technique that is commonly used to introduce antisense modulators into cultured cells includes electroporation. When LIPOFECTAMINE or LIPOFECTIN is used, the typical concentration range of antisense modulators administered to cultured cells is 1 nM-300 nM. When electroporation is used, the typical concentration range of antisense modulators administered to cultured cells is 625 nM-20,000 nM.

Following administration of antisense modulators to cultured cells, the cells are typically assayed 16-72 hours post-treatment. The cultured cells can be fixed, stained with antibodies and observed by microscopy to measure phenotypes such as cell survival, cell morphology and the levels of TDP-43 aggregates in the cytoplasm. The cultured cells can also be harvested to measure the levels of RNA or protein levels of target nucleic acids using methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments. The concentration of antisense modulator used can vary from cell line to cell line. Methods to determine the optimal concentrations of an antisense modulator for a particular cell line are well known in the art.

Methods for in vitro testing of modulators, including antisense modulators, are described in U.S. Pat. No. 10,577,604, which along with references cited therein, are incorporated herein in their entirety. Other modifications are known to those skilled in the art and are considered to be included in the embodiments herein.

In Vivo Testing of Antisense Modulators

In certain embodiments, the antisense modulators described herein can be administered to animals (all of references of which can include humans) in vivo to evaluate their safety, effects on the expression or activity of target gene(s), and effects on other phenotypes, such as, for example, survival, motor function, respiration, behavior, body weight, etc. In certain embodiments, the antisense modulators described herein can be administered to animals in vivo to evaluate the effects of antisense modulators on FNIP1 and/or FNIP2 expression or activity. In certain embodiments, the antisense modulators provided herein can be administered to animals in vivo to evaluate the safety of the compounds. In certain embodiments, the antisense modulators can be administered to animals in vivo to evaluate the effects of antisense modulators on one or more phenotypes, such as, for example, survival, motor function, respiration, behavior, body weight, etc. In certain embodiments, other modulators described herein, such as, for example, other oligonucleotide modulators, antibody modulators, peptide modulators, or small molecule modulators can be administered to animals in vivo to evaluate their effects on FNIP1 and/or FNIP2 expression or activity, or on one or more phenotypes, such as, for example, survival, motor function, respiration, behavior, body weight, etc. Methods to measure motor function are well known in the art and include, for example, the grip strength assay, rotarod assay, walking initiation analysis, balance beam test, pole climb assay, open field performance and hindpaw footprint tests. Methods to measure respiration are well known in the art and include, for example, whole body plethysmograph, invasive resistance, and compliance measurements in the animal. In some embodiments, testing can be performed in healthy animals. In other embodiments, testing can be performed in disease animals.

In certain embodiments, modulators described herein, including antisense modulators, are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline, for administration to animals. Administration includes parenteral routes of administration, such as, for example, intrathecal, intraperitoneal, intravenous, and subcutaneous, etc. Methods to calculate appropriate dosages of antisense modulators and dosing frequency are well known in the art and depends upon factors such as animal body weight and route of administration. Following the administration of modulators, including antisense modulators, to the animal, the animals are monitored at defined timepoints for the expression levels of target gene(s) such as FNIP1 and/or FNIP2, and effects on other phenotypes, such as, for example, survival, motor function, respiration, behavior, body weight, etc. The levels of FNIP1 and/or FNIP2 RNA or FNIP1 and/or FNIP2 protein can be measured in different tissues from the animal, such as, for example, the CSF, plasma, brain, spinal cord, lung, liver, kidney etc., using methods known in the art and described herein.

Methods for in vivo testing of modulators, including antisense modulators, are described in U.S. Pat. No. 10,577,604, which along with each of the references cited therein, are incorporated herein in their entirety. Other modifications are known to those skilled in the art and are considered to be included in the embodiments herein.

Other Modulators

In certain embodiments, provided herein are modulators other than antisense modulators, for example other oligonucleotide modulators (e.g., ribozyme, deoxyribozyme, or aptamers), antibody modulators, peptide modulators, small molecule modulators, and nucleic acid vectors, which can be administered to a cell, an animal, or a human subject, to modulate the expression or activity of a target polypeptide or nucleic acid. In some embodiments, provided herein are modulators other than antisense modulators, for example other oligonucleotide modulators (e.g., ribozyme, deoxyribozyme, or aptamers), antibody modulators, peptide modulators, small molecule modulators, and nucleic acid vectors, which can be administered to a cell, an animal, or a human subject, to reduce or inhibit the expression or activity of FNIP1 and/or FNIP2, in order to treat, prevent or ameliorate a disease, particularly neuromuscular or neurodegenerative diseases, such as for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), or other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. In other embodiments, provided herein are modulators other than antisense modulators, for example other oligonucleotide modulators (e.g., ribozyme, deoxyribozyme, or aptamers), antibody modulators, peptide modulators, small molecule modulators, and nucleic acid vectors, which can be administered to a cell, an animal, or a human subject, to increase the expression or activity of FNIP1 and/or FNIP2, in order to treat, prevent or ameliorate a disease such as an inflammatory disease, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers such as fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas (slow-growing tumors of the central nervous system), pancreatic neuroendocrine tumors, pheochromocytomas (noncancerous tumors of the adrenal glands), endolymphatic sac tumors, kidney cysts, lung cysts, and other diseases that are linked to loss-of-function of FNIP1 and/or FNIP2.

Gene Therapy

In certain embodiments, provided herein are compositions and methods involving transfer of genetic material to a cell, an animal, or a human subject, in order to treat, prevent, or ameliorate a disease, otherwise referred to as gene therapy. The genetic material can be integrated into the genome (e.g., via an integrating vector or by homology-directed repair). In certain embodiments, the genetic material is not integrated into the genome (e.g., carried on a non-integrating vector). The genetic material can be administered in vivo (directly into the patient), or the genetic material can be administered ex vivo (to cultured cells taken from the patient that are subsequently transplanted back into the patient). In certain embodiments, lentiviral vectors are used for ex vivo transfer of genetic material into hematopoietic and other stem cells. In other embodiments, adeno-associated viral (AAV) vectors are used for in vivo transfer of genetic material into postmitotic cell types. In some embodiments, AAV2 or AAV9 vectors are used for in vivo transfer of genetic material into the central nervous system (CNS). Compositions and methods of gene therapy as a therapeutic method are described by Anguela and High (Anguela and High, Annual Review of Medicine, 70, 273-288 (2019)), which along with references cited therein, are incorporated herein by reference in their entirety.

One goal of gene therapy is gene augmentation, which seeks to restore normal cellular function by increasing the expression or activity of a gene. In some embodiments, if a mutation in a gene leads to a loss-of-function of the polypeptide or nucleotide sequence encoded by the gene, which leads to the disease, a modulator or additional normal functional copies of the gene can be supplied to the cell to increase the expression of the gene, in order to treat, prevent or ameliorate the disease. In some embodiments, even if the disease is not caused by, or associated with, a loss-of-function of the polypeptide or nucleotide sequence encoded by the gene, gene augmentation can still be useful to treat, prevent or ameliorate the disease. Another goal of gene therapy is gene suppression, which seeks to restore cellular function by reducing the expression or activity of a gene. In some embodiments, if a mutation in a gene leads to a gain-of-function of the polypeptide or nucleotide sequence encoded by the gene, which leads to the disease, a modulator to suppress the expression or activity of the gene can be supplied to the cell to treat, prevent or ameliorate the disease. In some embodiments, even if the disease is not caused by, or associated with, a gain-of-function of the polypeptide or nucleotide sequence encoded by the gene, gene suppression can still be useful to treat, prevent or ameliorate the disease.

In certain embodiments, gene therapy comprises genome editing, which is the modification of the genome of the cell, for example, by removing pathogenic mutations or introducing beneficial mutations to one or more genetic features, in order to restore normal cellular function. In one embodiment, genome editing relies on an enzyme or enzyme complex, such as TALENs, CRISPR/Cas, zinc-finger nucleases (ZFNs), meganucleases, or other endonuclease system. The enzyme or enzyme complexes used for genome editing described here are non-exhaustive and other enzyme or enzyme complexes used for genome editing are well known to a person of ordinary skill in the art and are included in various embodiments. For example, genome editing as a therapeutic approach is described by Ho et al. (Ho B. X. et al. International Journal of Molecular Sciences 19: 2721 (2018)), which along with references cited therein, are incorporated herein by reference in their entirety.

The typical modus operandi of genome editing involves the inserting, replacing, or deleting of certain nucleotide sequences in the genome of an organism, often through the introduction of an enzyme or enzyme complex and an exogenous nucleotide sequence. In some embodiments, the enzyme or enzyme complex facilitates the genome editing process by creating site-specific double-stranded breaks in the genome. In certain embodiments, homology directed repair, such as homologous recombination, is used by the cell to replace parts of the genome using an exogenous sequence of nucleotides, which is often introduced into the cell via a nucleic acid vector. Homology directed repair uses the natural enzymatic mechanisms within the cell to repair double-stranded breaks in the genome through the use of a homologous template. By introducing an exogenous sequence of nucleotides comprising the desired modified nucleotide sequence flanked by nucleotide sequences that are homologous to the genomic nucleic acid sequence at or around the double-stranded break, the cell can utilize this exogenous sequence of nucleotides as the homologous template for the homology directed repair process, thereby inserting, modifying, or deleting the original sequence of nucleotides present at or around the double-stranded break. In other embodiments, non-homologous end-joining is used by the cell to directly repair double-stranded breaks in the genome without using a homologous template. During the non-homologous end-joining process, insertions or deletions are introduced into the genome, which in the case of protein-coding genes often leads to a change in the reading frame or introduction of a premature stop codon, thus rendering the gene non-functional.

In one embodiment, two double-stranded breaks are introduced at the region where genome editing is desired to increase the efficiency of homology-directed repair or non-homologous end-joining. In another embodiment, more than one genomic region is targeted for homology-directed repair or non-homologous end-joining by introducing more than one double-stranded break simultaneously at different genomic locations in the cell. In yet another embodiment, a single-stranded break (that is similarly capable of stimulating homology-directed repair or non-homologous end-joining) is introduced in the genome using an engineered endonuclease, instead of a double-stranded break, in order to reduce toxicity to the cell. In certain embodiments, genome editing can be achieved without single-stranded breaks, double-stranded breaks or using a homologous template. This can be achieved by using a catalytically impaired endonuclease (e.g., Cas9) fused to an engineered reverse transcriptase, which is programmed with a prime editing guide RNA (pegRNA) that both encodes the desired edits and specifies the target site, otherwise known as prime editing. Prime editing is described by Anzalone et al. (Anzalone et al., Nature, 576 (7785), 149-157 (2019)), which along with references cited therein, are incorporated herein by reference in their entirety.

In certain embodiments, genome editing involves making integrative changes (e.g., insertions, deletions or modifications) to the DNA sequence in the chromosome of the cell. This can be achieved by using an endonuclease that can recognize and cleave DNA sequences, for example, Cas9 and Cas12a (also known as Cpf1). In other embodiments, genome editing involves making non-integrative changes (e.g., insertions, deletions or modifications) to the RNA sequence in the cell. This can be achieved by using an endonuclease that can recognize and cleave RNA sequences, for example, Cas13. RNA-targeting CRISPR-Cas endonucleases and systems are described by Burmistrz et al. (Burmistrz et al., Int. J. Mol. Sci. 21, 1122 (2020)), which along with references cited therein, are incorporated herein by reference in their entirety.

In another embodiment, genome editing can comprise the correction of at least one point mutation using an engineered endonuclease that is catalytically inactive but able to recognize and bind to a specific sequence of DNA containing the point mutation, wherein the engineered endonuclease is coupled to a base editing enzyme, and correcting the point mutation via the activity of the coupled base editor. In one embodiment, the catalytically inactive endonuclease does not introduce a double-stranded break. Base editing can be used to introduce any of the four transition mutations, C to T, G to A, A to G, and T to C. Specifically, for DNA, the cytosine base editor (CBE) can alter a C-G base pair into a T-A base pair, while the adenine base editor (ABE) can alter an A-T base pair into a G-C pair. In another embodiment, genome editing can comprise the correction of at least one point mutation using an engineered endonuclease that is catalytically inactive but able to recognize and bind to a specific sequence of RNA containing the point mutation. For RNA, the RNA base editor (RBE) can convert adenine (A) to inosine (I). CRISPR/Cas-mediated base editing is described by Molla and Yang (Molla K. A. and Yang Y., Trends in Biotechnology, 37(10), 1121-1142, (2019)), and Grunewald et al. (Grunewald et al., Nature Biotechnology, 38, 861-864 (2020)), which along with references cited therein, are incorporated herein by reference in their entirety.

In yet another embodiment, genome editing can comprise using a catalytically inactive endonuclease linked to at least one epigenetic modification enzyme to effect a change to the epigenetic state of the genome. For example, catalytically inactive Cas9 (CRISPR endonuclease) can be coupled to epigenetic modification enzymes including but not limited to KRAB, DNMT3A, LSD1, p300, TET, VP64, SunTag-epieffector, SAM-epieffector, and the like. In one embodiment, the catalytically inactive endonuclease is coupled to activator and/or repressor domains to effect a change in the expression of at least one target gene. Some examples that are known to a skilled person in the art include CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). CRISPR/Cas-mediated epigenetic methods are described in Xie et al. (Xie et al., Stem Cells International, Article ID 7834175 (2018)), which along with references cited therein, are incorporated herein by reference in their entirety.

In certain embodiments, provided herein are compositions and methods of gene therapy to modulate the expression or activity of FNIP1 and/or FNIP2. In a preferred embodiment, provided herein are compositions and methods of gene therapy to decrease or inhibit the expression or activity of FNIP1 and/or FNIP2. In some embodiments, provided herein are compositions and methods of gene therapy to decrease the expression or activity of FNIP1 and/or FNIP2, in order to either reduce the levels of TDP-43 aggregates in the cytoplasm, or increase the levels of functional TDP-43 in the nucleus, or achieve a combination of both, thereby treating, preventing or ameliorating a disease, particularly a neuromuscular or neurodegenerative disease, such as ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), or other TDP-43 proteinopathies. In other embodiments, provided herein are compositions and methods of gene therapy to decrease the expression or activity of FNIP1 and/or FNIP2, in order to increase the activity of the HIF1α and/or VEGF pathways to promote angiogenesis, thereby treating, preventing or ameliorating a neuromuscular or neurodegenerative disease, such as ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), or other TDP-43 proteinopathy, as well as anemia or ischemic diseases such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. In some embodiments, provided herein are compositions and methods of gene therapy to decrease the expression or activity of FNIP1 and/or FNIP2, in order to decrease the expression or activity of FLCN, which can be used to treat, prevent or ameliorate diseases such as oxidative stress or obesity.

In yet other embodiments, provided herein are compositions and methods of gene therapy to increase the expression or activity of FNIP1 and/or FNIP2. In some embodiments, provided herein are compositions and methods of gene therapy to increase the expression or activity of FNIP1 and/or FNIP2, in order to decrease the activity of pro-inflammatory pathways, thereby treating, preventing or ameliorating inflammatory diseases. In other embodiments, provided herein are compositions and methods of gene therapy to increase the expression or activity of FNIP1 and/or FNIP2, in order to rescue the loss-of-function in the FNIP, FLCN or VHL pathways, thereby treating, preventing or ameliorating a disease such as von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers such as fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas, pancreatic neuroendocrine tumors, pheochromocytomas, endolymphatic sac tumors, kidney cysts, lung cysts, and other diseases that are linked to loss-of-function of FNIP1 and/or FNIP2. In one embodiment, genome editing is used to insert, delete, or modify DNA sequences associated with FNIP1 and/or FNIP2, such as sequences described by SEQ ID NOs: 1-15. In one embodiment, genome editing is used to insert, delete or modify RNA sequences associated with FNIP1 and/or FNIP2, such as sequences described by SEQ ID NOs: 1-15. Genome editing via enzymes or enzyme complexes and their methods of delivery can be produced by any number of methods known to a person who is skilled in the art, which are incorporated herein (see below for specific methods).

Nucleic Acid Vector

In one embodiment, the modulator comprises a nucleic acid vector. In one embodiment, the nucleic acid vector can encode for the modulator of choice, or a nucleobase sequence that includes the modulator of choice, or a nucleobase sequence complementary to the modulator of choice, wherein the modulator of choice is, for example, a siRNA, miRNA, lncRNA, gRNA, an antisense oligonucleotide, or a gene. The modulator, or a nucleobase sequence containing the modulator, or a nucleobase sequence complementary to the modulator, can be expressed following delivery of the nucleic acid vector into a target cell. The terms vector and nucleic acid vector herein are used interchangeably.

In some embodiments, the nucleic acid vector encodes for one or more functional copies of the gene of interest, such as FNIP1 and/or FNIP2. The additional copies of FNIP1 and/or FNIP2 are expressed in the cell to increase the levels of FNIP1 and/or FNIP2 in the cell. In certain embodiments, the nucleic acid vector encodes for a modulator that is expressed to form an activator. The activator targets FNIP1 and/or FNIP2 nucleic acids or polypeptides to increase the expression or activity of FNIP1 and/or FNIP2. In some embodiments, the activator targets DNA sequences encoding FNIP1 and/or FNIP2, or DNA sequences that regulate the expression of FNIP1 and/or FNIP2, and increases transcription of the FNIP1 and/or FNIP2 gene to mRNA. In another embodiment, the activator targets the FNIP1 and/or FNIP2 mRNA transcribed from the gene and increases translation of FNIP1 and/or FNIP2 mRNA into the polypeptide. In a preferred embodiment, the nucleic acid vector encodes for a modulator that is expressed to form an inhibitor. The inhibitor targets the polypeptide of interest, such as FNIP1 and/or FNIP2, and inhibits or reduces the expression or activity of FNIP1 and/or FNIP2. In some embodiments, the inhibitor targets DNA sequences encoding FNIP1 and/or FNIP2, or DNA sequences that regulate the expression of FNIP1 and/or FNIP2, and inhibits or reduces transcription of the FNIP1 and/or FNIP2 gene to mRNA. In some embodiments, the inhibitor targets the FNIP1 and/or FNIP2 mRNA transcribed from the gene and inhibits or reduces translation of FNIP1 and/or FNIP2 mRNA into the polypeptide.

In another embodiment, the nucleic acid vector encodes for any of the modulator embodiments described herein. In certain embodiments, the nucleic acid vector is modified to enable a viral delivery method, such as pseudotyping, adaptor targeting, or genetic systems targeting, described herein. In other embodiments, the nucleic acid vector is modified to enable a non-viral delivery method described herein. In one embodiment, the nucleic acid vector for non-viral delivery is a minimized DNA vector. In one embodiment, the minimized DNA vector lacks antibiotic resistance genes that are typically present in plasmid DNA vectors. Minimized DNA vectors have the advantages of high transfection efficiency and high production yields over regular plasmid DNA. Furthermore, the lack of antibiotic resistance genes helps to reduce the risk of spread of antibiotic resistance genes in the environment. In one embodiment, the minimized DNA vector is a minicircle, wherein sequences of bacterial origin such as the origin of replication are removed. In one embodiment, the minimized DNA vector is a minivector, which can be smaller than a minicircle. Various advances in the design of non-viral minimized DNA vectors, as well as methods of production and use are described in Hardee et al. (Hardee et al., Genes, 8, 65 (2017)), which together with references cited therein, are incorporated herein in their entirety. Nucleic acid vectors and their methods of delivery (e.g., modified virus) can be produced by any number of methods known to a person who is skilled in the art (see below for specific methods).

Ribozyme or Deoxyribozyme

In some embodiments, the modulator can be a therapeutic ribozyme or deoxyribozyme.

A ribozyme or deoxyribozyme in various embodiments can be in any format well known to a person skilled in the art. Ribozymes and deoxyribozymes are sequences of nucleotides (e.g., RNA and DNA sequences, respectively) with enzymatic properties. Ribozymes and deoxyribozymes have been developed to target other molecules, such as RNA introduced by viruses. The enzymatic properties of ribozymes and deoxyribozymes can be used to catalyze, for example, the ligation or cleavage of RNA or DNA via hydrolysis or transesterification of the phosphate groups of the RNA or DNA molecule. Other uses of ribozymes and deoxyribozymes can include catalysis of peptide bonds, as is commonly found within ribosomes. In some cases, the ribozyme or deoxyribozyme activity can be catalyzed by the presence of one or more metal ions. In certain cases, ribozymes or deoxyribozymes can have the ability to self-synthesize or self-splice. A more detailed description of ribozymes and their therapeutic applications is given by Mulhbacher et al. (Mulhbacher et al., Current Opinion in Pharmacology, 10:551-556 (2010)). The Mulhbacher et al. reference, and references cited therein, are incorporated herein by reference in its entirety.

In some embodiments, a ribozyme or deoxyribozyme modulator is used to modulate the translation of mRNA of a target genetic sequence, such as FNIP1 and/or FNIP2. In some embodiments, a ribozyme or deoxyribozyme modulator is used to inhibit or decrease the expression or activity of FNIP1 and/or FNIP2. In other embodiments, a ribozyme or deoxyribozyme modulator is used to increase the expression or activity of FNIP1 and/or FNIP2.

In certain embodiments, a ribozyme or deoxyribozyme modulator is chemically attached to a large molecule or scaffold, creating a modulator-scaffold molecular complex. The modulator-scaffold molecular complex can enable additional functionality such as increased activity or efficacy of the modulator's enzymatic activity, improve the modulator's half-life and stability, or enable detection, tracing, or other diagnostic or therapeutic functionality.

In other embodiments, a ribozyme or deoxyribozyme modulator is chemically modified to increase activity or efficacy of the modulator's enzymatic activity, improve the modulator's half-life and stability, or enable detection, tracing, or other diagnostic or therapeutic functionality. In one embodiment, a ribozyme or deoxyribozyme modulator is chemically modified in the 2′ position of its constituent ribose.

Ribozymes or deoxyribozymes can be produced by any number of methods known to a person who is skilled in the art (see below for specific methods).

Aptamer

In one embodiment, the modulator can be a therapeutic aptamer. A therapeutic aptamer can be an oligonucleotide aptamer, an oligopeptide aptamer, or a polypeptide aptamer.

Aptamers in various embodiments can be in any format well known to a person skilled in the art. Aptamers are oligonucleotide, oligopeptide, or polypeptide molecules engineered to have binding specificity to a target molecule of choice, often through the influence of higher-level structural factors.

In certain embodiments, the aptamer modulator is integrated into a larger nucleotide or peptide scaffold. The scaffold can enable additional functionality such as increased activity or efficacy of the modulator, promoting an immunological response, detection or tracing, increased half-life and stability, or other diagnostic or therapeutic functionality. In some embodiments, the scaffold is a peptide comprising one of the following: a monobody, an anticalin, a polypeptide with a Kunitz domain, an avimer, a knottin, a fynomer, or an atrimer.

In other embodiments, the aptamer modulator is integrated into a ribozyme, deoxyribozyme, or enzyme to form an aptamer-zyme complex. The aptamer-zyme complex can have additional functionality or specificity towards a targeted polypeptide or nucleotide sequence.

In some embodiments, the aptamer is an oligonucleotide chemically modified to increase activity or efficacy of the modulator's enzymatic activity, improve the modulator's half-life and stability, detection or tracing, or other diagnostic or therapeutic functionality. A list of common chemical modifications in oligonucleotide aptamers is described by Dunn et al. (Dunn et al., Nature Reviews Chemistry, 1(10):0076 (2017)). The Dunn et al. reference, and references cited therein, is incorporated by reference in its entirety and is known to a person of ordinary skill in the art.

In certain embodiments, a therapeutic aptamer is used to modulate the expression or activity of a target genetic sequence or protein, such as FNIP1 and/or FNIP2. In some embodiments, a therapeutic aptamer is used to inhibit or decrease the expression or activity of FNIP1 and/or FNIP2. In other embodiments, a therapeutic aptamer is used to increase the expression or activity of FNIP1 and/or FNIP2. Aptamers can be produced by any number of methods known to a person who is skilled in the art (see below for specific methods, such as in vitro selection).

Antibody and Related Protein

In one embodiment, the modulator can be a therapeutic protein or polypeptide. A therapeutic protein can be an antibody, antibody fragment, or monobody. The terms “peptide”, “protein” and “polypeptide” herein are used interchangeably

Antibodies can be in any format well known to a person skilled in the art. Antibodies are heteromultimeric glycoproteins consisting of two larger polypeptide heavy chains and two smaller polypeptide light chains. Each of the heavy chains and light chains comprise a variable region and constant regions. The variable region of the light chain is aligned with the variable region of the heavy chain, and the constant regions of the light chain is aligned with the constant regions of the heavy chain. Each light chain is bound together to a heavy chain via a disulfide covalent bond, and the two heavy chains are bound together by disulfide covalent bonds that can vary in quantity depending on the type of antibody. Antibodies are typically grouped into five different isotypes in mammals: IgA, IgD, IgE, IgG, and IgM. These isotypes are determined by the amino acid sequence of the constant regions of the heavy chains, wherein within each isotype the constant regions of the heavy chains are identical. Light chains are grouped into two different type in mammals: kappa and lambda. Only one type of light chain is present in each antibody, except in the case when the antibody is bispecific. The variable region of the heavy and light chains of the antibody confer the antibody's ability to bind to specific antigens, which are otherwise known as the complementarity-determining region (CDR). The CDR is defined by Dondelinger et al. (Dondelinger et al., Frontiers in Immunology, 9: 2278 (2018)), which along with references cited therein, are incorporated herein in its entirety. The different isotypes determined by the constant regions enable different crystallizable fragments to bind to the antibody or antibody-antigen complex. These crystallizable fragments are associated with different pathways in immunological response and other physiological effects such as clearance rate, cell-mediated cytotoxicity, phagocytosis through agglutination or precipitation, and complement-dependent cytotoxicity.

In a preferred embodiment, the antibody modulator contains a constant region of the IgG isotype derived from human sources. In another embodiment, the antibody can be humanized or chimeric. In a preferred embodiment, the antibody is monoclonal. In other embodiments, the antibody is polyclonal. In another embodiment, the antibody modulator is used in conjunction with other antibody modulators of other polypeptides or nucleotide sequence of interest to inhibit the expression or activity of multiple polypeptides and/or multiple nucleotide sequences within one or more functional pathways. In another embodiment, the antibody modulator is used in conjunction with other antibody modulators of other polypeptides or nucleotide sequence of interest to increase the expression or activity of multiple polypeptides and/or multiple nucleotide sequences within one or more functional pathways. In yet another embodiment, the antibody is bispecific, being capable of binding to two separate polypeptides or nucleotide sequences, or any combination thereof of interest.

In certain embodiments, the antibody can comprise additional polypeptide chains or functional groups that confer additional properties to the antibody, such as enhanced immune response, enhanced antigen specificity, or stability.

In certain embodiments, the modulator is an antibody fragment, such as a single-chain variable fragment or antigen binding fragment, which is able to bind to and inhibit the expression or activity of the polypeptide or nucleotide sequence of interest. In other embodiments, the modulator is an antibody fragment, such as a single-chain variable fragment or antigen binding fragment, which is able to bind to and increase the expression or activity of the polypeptide or nucleotide sequence of interest.

In some embodiments, the modulator is a monobody which is able to bind to and inhibit the expression or activity of the polypeptide or nucleotide sequence of interest. In other embodiments, the modulator is a monobody which is able to bind to and increase the expression or activity of the polypeptide or nucleotide sequence of interest. Monobodies in various embodiments can be in any format well known to a person skilled in the art. Monobodies are proteins that are smaller and less complex than antibodies, and that are engineered to have antigen-binding properties similar to that of antibodies. Monobodies are created with a fibronectin type III scaffold in which certain sections of its amino acid sequence is varied to create variable specificity to antigens of choice.

In certain embodiments, there is provided a modulator comprising an antibody, antibody fragment, monobody, or other peptide modulator that binds to the polypeptide or nucleotide sequence of interest, thus modulating the expression or activity of the target polypeptide or nucleotide sequence of interest. In other embodiments, there is provided a modulator comprising an antibody, antibody fragment, monobody, or other peptide modulator that binds to the polypeptide or nucleotide sequence of interest, thus modulating the interaction of the polypeptide or nucleotide sequence of interest with other molecules in the targeted functional pathway. In some embodiments, the modulator comprises an antibody, antibody fragment, monobody or other peptide modulator that binds to the FNIP1 and/or FNIP2 protein or nucleic acid, thus inhibiting or decreasing the expression or activity of FNIP1 and/or FNIP2. In some embodiments, the modulator comprises an antibody, antibody fragment, monobody, or other peptide modulator that disrupts the activity of FNIP1 and/or FNIP2, thereby preventing or reducing its ability to positively regulate the expression or activity of FLCN, thus resulting in a decrease in expression or activity of FLCN. In some embodiments, the antibody, antibody fragment, monobody, or other peptide modulator targets the longin and/or DENN domains of FNIP1 and/or FNIP2, thereby blocking its interaction with FLCN, thus preventing the formation of the FNIP1-FLCN complex and/or FNIP2-FLCN complex respectively. In yet other embodiments, the modulator comprises an antibody, antibody fragment, monobody, or other peptide modulator that decreases the expression or activity of FNIP1 and/or FNIP2, thereby resulting in a decrease in the levels of TDP-43 aggregates in the cytoplasm, or an increase in the levels of functional TDP-43 in the nucleus, or a combination of both.

In certain embodiments, the modulator comprises an antibody, antibody fragment, monobody, or other peptide modulator that binds to the FNIP1 and/or FNIP2 protein or nucleic acid, thus increasing the expression or activity of FNIP1 and/or FNIP2. In other embodiments, the modulator comprises an antibody, antibody fragment, monobody, or other peptide modulator that increases the activity of FNIP1 and/or FNIP2, thereby increasing its ability to positively regulate the expression or activity of FLCN, thus resulting in an increase in expression or activity of FLCN. In some embodiments, the antibody, antibody fragment, monobody, or other peptide modulator targets the longin and/or DENN domains of FNIP1 and/or FNIP2, thereby promoting its interaction with FLCN to increase the expression or activity of the FNIP1-FLCN complex and/or FNIP2-FLCN complex respectively. Examples of antibody modulators that target FNIP1 and/or FNIP2 are provided in Example 8.

In one embodiment, antibody modulators that are capable of targeting FNIP1 and/or FNIP2 can be obtained commercially from a variety of sources, such as described in the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.). In some embodiments, provided herein are antibodies, antibody fragments, monobodies, or other peptide modulators for the detection of human FNIP1 and/or FNIP2, as well as their homologs in other animals such as the mouse, rat, zebrafish, or monkey. Such antibodies are available from commercial sources, examples of which are provided in Table 14. In some embodiments, provided herein are antibodies, antibody fragments, monobodies, or other peptide modulators that bind to the same epitope as at least one antibody described in Table 14. In other embodiments, the antibody, antibody fragment, monobody, or other peptide modulator binds to a different epitope as that of the modulators described in Table 14. In some embodiments, the antibody, antibody fragment, monobody, or peptide modulator comprises a CDR that is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the CDR of at least one antibody described in Table 14, as assessed by sequence alignment or other scoring methods known in the art. Such protein sequence alignment or scoring methods can take into account the 3D structure or conformation of the CDR region. Protein sequence alignment and scoring methods are described by Wang et al. (Wang et al., BMC Bioinformatics, 19: 529 (2018)) and Kunik et al. (Kunik et al., PLoS Computational Biology, 8(2): e1002388 (2012)), which together with references cited therein, are incorporated herein in their entirety.

Antibodies, antibody fragments, monobodies, and other peptide modulators can be produced by any number of methods known to a person who is skilled in the art (see below for specific methods).

Small Molecule

In one embodiment, the modulator is a small molecule.

A small molecule in various embodiments can be in any format well known to a person skilled in the art. A small molecule is generally referred to as a molecule with molecular weight less than 900 daltons. In other embodiments, a small molecule refers to a molecule with molecular weight less than 1000, less than 2000, or alternatively less than 3000 daltons.

In certain embodiments, a small molecule modulator can bind to a target nucleic acid, wherein the target nucleic acid encodes FNIP1 and/or FNIP2. In a preferred embodiment, the target nucleic acid is an RNA transcript encoding FNIP1 and/or FNIP2. In other embodiments, the target nucleic acid is a DNA molecule encoding FNIP1 and/or FNIP2.

In certain embodiments, target nucleic acids can comprise nucleobase sequences encoding FNIP1 and/or FNIP2, including but not limited to the following: the reverse complement of RefSeq Accession No. NC_000004.12 truncated from nucleotides 158767000 to 158910000 (incorporated herein as SEQ ID NO: 1), RefSeq Accession No. NM_020840.3 (incorporated herein as SEQ ID NO: 2), RefSeq Accession No. NM_001323916.2 (incorporated herein as SEQ ID NO: 3), RefSeq Accession No. NM_001346043.2 (incorporated herein as SEQ ID NO: 4), RefSeq Accession No. NM_001366843.1 (incorporated herein as SEQ ID NO: 5), RefSeq Accession No. XM_017008487.1 (incorporated herein as SEQ ID NO: 6), RefSeq Accession No. XM_005263158.2 (incorporated herein as SEQ ID NO: 7), RefSeq Accession No. XM_005263160.3 (incorporated herein as SEQ ID NO: 8), RefSeq Accession No. XR_001741297.1 (incorporated herein as SEQ ID NO: 9), RefSeq Accession No. XM_024454161.1 (incorporated herein as SEQ ID NO: 10), RefSeq Accession No. NC_000005.10 truncated from nucleotides 131639000 to 131799000 (incorporated herein as SEQ ID NO: 11), RefSeq Accession No. NM_133372.3 (incorporated herein as SEQ ID NO: 12), RefSeq Accession No. NM_001008738.3 (incorporated herein as SEQ ID NO: 13), RefSeq Accession No. NM_001346113.2 (incorporated herein as SEQ ID NO: 14), and RefSeq Accession No. NM_001346114.2 (incorporated herein as SEQ ID NO: 15). In some embodiments, small molecule modulators can also target other nucleobase sequences encoding FNIP1 and/or FNIP2 (e.g. other DNA sequences, cDNA sequences, scaffolds, or mRNA transcript variants), including but not limited to entries that can be found by accession number in databases such as NCBI and GENBANK, and which are incorporated herein by reference. In other embodiments, nucleobase sequences encoding FNIP1 and/or FNIP2 include previous and future versions of nucleobase sequences encoding FNIP1 and/or FNIP2, which can be found by accession number in databases such as NCBI and GENBANK, and which are also incorporated herein by reference. In other embodiments, nucleobase sequences encoding FNIP1 and/or FNIP2 include mRNA transcripts that are formed from the utilization of alternative polyadenylation sites. The nucleobase sequence set forth in each SEQ ID NO contained herein is independent of any modification to a nucleobase, a sugar moiety, or an internucleoside linkage.

In certain embodiments, a small molecule modulator can bind to at least one target region within the target nucleic acid. A target region is a structurally defined region of the target nucleic acid. Examples of a target region include but are not limited to an exon, an intron, an exon-intron junction, an intron-exon junction, an exon-exon junction, a 3′ untranslated region (3′ UTR), a 5′ untranslated region (5′ UTR), a G-quadruplex, a translation initiation region, a translation termination region, a 5′ donor splice site, a 3′ acceptor splice site, a start codon, an upstream open reading frame (ORF), a repeat region, a hexanucleotide repeat expansion, a splice enhancer region, an exonic splicing enhancer (ESE), a splice suppressor region, an exonic splicing silencer (ESS), an intronic splicing enhancer (ISE), an intronic splicing silencer (ISS), an RNA destabilization motif, an RNA stabilization motif, a miRNA binding site, an RNA-binding protein (RBP) binding site, a stem-loop, a bulge, a hairpin, a junction, or other structurally defined nucleic acid region. In one embodiment, the target regions, or structurally defined regions for FNIP1 and/or FNIP2, can be obtained by accession number from sequence databases such as NCBI, UNIPROT and GENBANK, and such information is incorporated herein by reference. In other embodiments, the target regions, or structurally defined regions for FNIP1 and/or FNIP2, can be obtained using computational tools well known in the art that predict the existence of a target region, and/or that predict the stability or structure of a nucleic acid region.

The determination of suitable target regions can include a comparison of the nucleobase sequence of the target region to other sequences throughout the genome. For example, the BLAST algorithm, or other structure comparison algorithms, can be used to identify regions of similarity amongst different sequences. This comparison can enable the selection of target regions that can be specifically targeted by a small molecule modulator, with a corresponding reduced likelihood of the small molecule modulator binding in a non-specific manner to non-target or off-target sequences.

In some embodiments, targeting includes determination of at least one target region within a target nucleic acid to which a small molecule modulator can bind in order to produce a desired effect. The desired effect can be a decrease or increase in mRNA levels of a target nucleic acid. The desired effect can be a decrease or increase in the stability of mRNA of a target nucleic acid. The desired effect can also be a decrease or increase in levels of a protein encoded by the target nucleic acid. The desired effect can also be a phenotypic change associated with a change in mRNA levels of a target nucleic acid, and/or change in protein levels encoded by the target nucleic acid. In certain embodiments, the desired effect of using a small molecule modulator to target at least one target region within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it binds, is a reduction in FNIP1 and/or FNIP2 mRNA levels. In other embodiments, the desired effect of using a small molecule modulator to target at least one target region within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it binds is a reduction in FNIP1 and/or FNIP2 protein levels. In yet other embodiments, the desired effect of using a small molecule modulator to target at least one target region within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it binds is a phenotypic change associated with the reduction of FNIP1 and/or FNIP2 mRNA or protein levels. In certain embodiments, the desired effect of using a small molecule modulator to target at least one target region within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it binds, is an increase in FNIP1 and/or FNIP2 mRNA levels. In other embodiments, the desired effect of using a small molecule modulator to target at least one target region within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it binds is an increase in FNIP1 and/or FNIP2 protein levels. In yet other embodiments, the desired effect of using a small molecule modulator to target at least one target region within a target nucleic acid encoding FNIP1 and/or FNIP2 to which it binds is a phenotypic change associated with the increase of FNIP1 and/or FNIP2 mRNA or protein levels.

In certain embodiments, the small molecule modulators described herein can bind to any target nucleic acid comprising nucleotide sequences encoding FNIP1 and/or FNIP2. In some embodiments, the small molecule modulators can bind to target nucleic acids at any stage of RNA processing within the cell, for example, pre-mRNA or mature mRNA. In yet other embodiments, small molecule modulators can bind to any target region(s) within the target nucleic acid, including but not limited to an exon, an intron, an exon-intron junction, an intron-exon junction, an exon-exon junction, a 3′ untranslated region (3′ UTR), a 5′ untranslated region (5′ UTR), a G-quadruplex, a translation initiation region, a translation termination region, a 5′ donor splice site, a 3′ acceptor splice site, a start codon, an upstream open reading frame (ORF), a repeat region, a hexanucleotide repeat expansion, a splice enhancer region, an exonic splicing enhancer (ESE), a splice suppressor region, an exonic splicing silencer (ESS), an intronic splicing enhancer (ISE), an intronic splicing silencer (ISS), a RNA destabilization motif, a RNA stabilization motif, a miRNA binding site, an RNA-binding protein (RBP) binding site, a stem-loop, a bulge, a hairpin, a junction, or other structurally defined nucleic acid region. In one embodiment, small molecule modulators can bind to at least one exon present in SEQ ID NOs: 1-15. In other embodiments, small molecule modulators can bind to target regions other than exons that are present in SEQ ID NOs: 1-15, wherein such regions can be described in databases such as NCBI, UNIPROT and GENBANK, which are incorporated herein by reference. In other embodiments, small molecule modulators can bind to target regions other than exons that are present in SEQ ID NOs: 1-15, wherein such regions can be predicted using computational tools well known in the art that predict the presence of a target region, and/or that predict the stability or structure of a nucleic acid region.

In certain embodiments, the small molecule modulators described herein can bind to all RNA transcript variants of FNIP1 and/or FNIP2. In other embodiments, the small molecule modulators described herein bind selectively to at least one RNA transcript variant of FNIP1 and/or FNIP2. Transcript variants of FNIP1 and/or FNIP2 can include mRNA transcripts generated by differential splicing, or mRNA transcripts containing mutations (e.g., SNPs, INDELs etc.) when compared to a reference sequence. In certain embodiments, the small molecule modulators described herein inhibit the expression of all transcript variants of FNIP1 and/or FNIP2. In certain embodiments, the small molecule modulators described herein inhibit expression of all transcript variants of FNIP1 and/or FNIP2 equally. In certain embodiments, the small molecule modulator described herein preferentially inhibits the expression of certain transcript variants of FNIP1 and/or FNIP2. In other embodiments, the small molecule modulators described herein increase the expression of all transcript variants of FNIP1 and/or FNIP2. In certain embodiments, the small molecule modulators described herein increase the expression of all transcript variants of FNIP1 and/or FNIP2 equally. In certain embodiments, the small molecule modulator described herein preferentially increases the expression of certain transcript variants of FNIP1 and/or FNIP2. In certain embodiments, small molecule modulators described herein are useful for reducing cytoplasmic TDP-43 aggregates, or increasing the levels of functional TDP-43 in the nucleus, or a combination thereof. In certain embodiments, small molecule modulators described herein are useful for normalizing the expression of various mis-regulated genes.

Thus, in certain embodiments, provided herein are modulators comprising a small molecule that binds to a nucleic acid encoding FNIP1 and/or FNIP2, thus inhibiting or reducing the expression of FNIP1 and/or FNIP2. In one embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and inhibits its transcription. In one embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and degrades or destabilizes it. In one embodiment, the small molecule modulator targets a region of a nucleic acid encoding FNIP1 and/or FNIP2 that is an RNA stabilization site, thus destabilizing the nucleic acid and reducing its expression levels. In one embodiment, the small molecule modulator targets a region of a nucleic acid encoding FNIP1 and/or FNIP2 that is an RBP binding site for a stabilization factor, thus destabilizing the nucleic acid and reducing its expression levels. In one embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and inhibits its translation. In yet another embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and modulates its splicing, thereby decreasing the expression or activity of FNIP1 and/or FNIP2. In some embodiments, the small molecule modulator is a bivalent compound that is capable of binding to both a nucleic acid encoding FNIP1 and/or FNIP2 and a ribonuclease such as RNase L to induce degradation of the nucleic acid encoding FNIP1 and/or FNIP2. Such bivalent compounds are known as RIBOTACs (ribonuclease-targeting chimeras) and are described by Dey et al. (Dey et al., Cell Chemical Biology, 26(8): 1047-1049 (2019)), which together with references cited therein, are incorporated herein in their entirety.

In other embodiments, the modulator comprises a small molecule that binds to a nucleic acid encoding FNIP1 and/or FNIP2, thus increasing the expression of FNIP1 and/or FNIP2. In one embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and increases its transcription. In one embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and stabilizes it. In one embodiment, the small molecule targets a region of a nucleic acid encoding FNIP1 and/or FNIP2 that is an RBP binding site for a destabilization factor, thus stabilizing the nucleic acid and increasing its expression levels. In one embodiment, the small molecule targets a region of a nucleic acid encoding FNIP1 and/or FNIP2 that is a miRNA binding site, thus increasing the stability and expression of the nucleic acid. In one embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and increases its translation. In yet another embodiment, the small molecule modulator targets a nucleic acid encoding FNIP1 and/or FNIP2 and modulates its splicing, thereby increasing the expression or activity of FNIP1 and/or FNIP2.

In certain embodiments, there is provided a modulator comprising a small molecule that binds to a polypeptide or protein of interest, thus modulating the expression or activity of the target polypeptide or protein of interest. In other embodiments, there is provided a modulator comprising a small molecule that binds to a polypeptide or protein of interest, thus modulating the interaction of the polypeptide or protein of interest with other molecules in the targeted functional pathway. In one embodiment, the modulator comprises a small molecule that binds to a FNIP1 and/or FNIP2 protein, thus inhibiting or reducing the activity of FNIP1 and/or FNIP2 protein. In some embodiments, the modulator comprises a small molecule that decreases the expression or activity of FNIP1 and/or FNIP2 protein, thereby preventing or reducing its ability to positively regulate the expression or activity of FLCN, thus resulting in a decrease in expression or activity of FLCN. In some embodiments, the small molecule modulator targets the longin and/or DENN domains of FNIP1 and/or FNIP2 protein, thereby blocking its interaction with FLCN, thus preventing the formation of the FNIP1-FLCN complex and/or FNIP2-FLCN complex respectively. In yet other embodiments, the modulator comprises a small molecule that decreases the expression or activity of FNIP1 and/or FNIP2 protein, thereby resulting in a decrease in the levels of TDP-43 aggregates in the cytoplasm, or an increase in the levels of functional TDP-43 in the nucleus, or a combination of both. In certain embodiments, the small molecule modulator reduces or inhibits the expression or activity of FNIP1 and/or FNIP2 protein by targeting it for degradation in the cell. In some embodiments, the small molecule modulator is a bivalent compound that is capable of binding to both FNIP1 and/or FNIP2 and an E3 ubiquitin ligase to induce ubiquitination of FNIP1 and/or FNIP2 and its subsequent degradation by the proteasome. Such bivalent compounds are known as PROTACS (proteolysis-targeting chimeras) and are described by Toure et al. (Toure et al., Angew. Chem. Int. Ed., 55: 1966-1973 (2016)), which together with references cited therein, are incorporated herein in their entirety.

In other embodiments, the modulator comprises a small molecule that binds to an FNIP1 and/or FNIP2 protein, thus increasing the activity of FNIP1 and/or FNIP2 protein. In some embodiments, the modulator comprises a small molecule that increases the expression or activity of FNIP1 and/or FNIP2 protein, thereby increasing its ability to positively regulate the expression or activity of FLCN, thus resulting in an increase in expression or activity of FLCN. In some embodiments, the small molecule modulator targets the longin and/or DENN domains of FNIP1 and/or FNIP2 protein, thereby promoting its interaction with FLCN, thus increasing the formation of the FNIP1-FLCN complex and/or FNIP2-FLCN complex respectively. In another embodiment, provided herein are bivalent small molecule modulators that are capable of increasing the interaction of FLCN with FNIP1 or FNIP2, thereby leading to an increase in the levels or activity of the FNIP1-FLCN and/or FNIP2-FLCN complex respectively.

Novel small molecule modulators that target the FNIP1 and/or FNIP2 nucleic acid and/or protein are provided in Example 7. In certain embodiments, provided herein are small molecule modulators comprising at least one exemplar described in Table 12 and/or Table 13. In certain embodiments, the small molecule modulator comprises at least one scaffold described in Table 12 and/or Table 13. In some embodiments, the small molecule modulator, or part thereof, is similar to at least one exemplar or scaffold in Table 12 and/or Table 13. Similarity between small molecules can be determined using methods well known in the art, such as for example, deriving the Tanimoto index, Dice index, Cosine coefficient or Soergel distance etc. Such methods are described in Bajusz et al. (Bajusz et al., Journal of Cheminformatics, 7: Article number 20 (2015)), which together with the references cited therein, are incorporated herein in their entirety. In some embodiments, provided herein are small molecule modulators, or part thereof, which have a Tanimoto index of at least 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00 compared to at least one exemplar or scaffold described in Table 12 and/or Table 13. The predicted dissociation constants of each small molecule exemplar, as calculated using software known in the art, are also described under the column “Exemplar Dissociation Constants” in Table 12 and Table 13. Small molecule modulators with smaller dissociation constants are predicted to bind more strongly to the target nucleic acid and/or protein.

Small molecules can be produced by any number of methods known to a person who is skilled in the art (see below for specific methods).

Pharmaceutical Compositions

A set of embodiments provides a pharmaceutical composition comprising at least one modulator together with a pharmaceutically acceptable carrier or diluent. The carrier or diluent of the pharmaceutical composition must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof. The pharmaceutical composition may be in unitary dosage form suitable, in particular, for administration orally, rectally, percutaneously, by parenteral injection or by inhalation. In some cases, administration can be via intravenous injection. For example, in preparing the composition in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions containing the modulators described herein may be formulated in oil for prolonged action. Appropriate oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long chain fatty acids and mixtures of these and other oils. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired composition. The composition may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.

Pharmaceutical compositions can be created using standard practices that are known to a person who is skilled in the art. Pharmaceutical compositions can be designed for administration in one of a number of various methods that are known to a person who is skilled in the art. A more detailed list of common practices is described by Wu & Chen (US 2018/0112272 A1), which along with references cited therein, are incorporated herein in its entirety.

In one embodiment, the pharmaceutical composition is for parenteral administration. In certain embodiments, compositions for parenteral administration can be sterile solutions, emulsions or suspensions that can be prepared from a solid or lyophilized form prior to administration. In other embodiments, the composition can contain certain adjuvants, anesthetics, buffering agents, or wetting agents that promote more effective distribution of the composition, facilitate ease of administration of the composition, or improve patient response or wellbeing. In one embodiment, the pharmaceutical composition is for intrathecal administration. In other embodiments, the pharmaceutical composition is for intramuscular, intracerebral, intracerebroventricular, intravenous, intravitreal or intraocular administration.

In another embodiment, the pharmaceutical composition is for gastrointestinal or enteric administration. In one embodiment, the pharmaceutical composition can be administered orally. In certain embodiments, compositions for gastrointestinal or oral administration can be a tablet, powder, capsule, or liquid. Such compositions can be formulated with a solid or liquid physiologically compatible carrier, including but not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate. In some embodiments, compositions can be formulated with disintegrants, including but not limited to starches, clays, celluloses, aligns, gums, and polymers, to facilitate the dissolution of solids. In other embodiments, compositions can also be formulated with lubricants, including but not limited to silicon dioxide, talc, or stearic acids, to facilitate the effective manufacturing of the composition.

In another embodiment, the pharmaceutical composition is administered transdermally or topically, such as in the form of an ointment, cream, or gel. In another embodiment, the pharmaceutical composition is administered transmucosally, such as in the form of a spray or a suppository.

In one embodiment, the pharmaceutical composition can be administered by nasal administration, including but not limited to, an inhalant, or delivered in an aerosol delivery device, such as an atomizer, nebulizer, or vaporizer. The aerosol delivery devices mentioned herein and other aerosol delivery devices are well known to a person of ordinary skill in the art and are included in various embodiments herein.

In another embodiment, the pharmaceutical composition is delivered via a targeted method that introduces or directs the pharmaceutical composition directly to the affected cells. Manish and Vimukta (Manish and Vimukta, Research Journal of Chemical Sciences, 1(2), 135-138 (2011)) describe common methods for targeted drug delivery, which along with references cited therein, are incorporated herein by reference in its entirety.

One set of embodiments provide for pharmaceutical compositions and methods, comprising a modulator and a pharmaceutically acceptable carrier or diluent mentioned herein, which can be administered to a cell, an animal, or a human subject to inhibit or decrease the expression or activity of FNIP1 and/or FNIP2 in the cell, animal or human subject. Such treatment methods can be used to treat, ameliorate, or prevent diseases, particularly neuromuscular or neurodegenerative diseases, such as for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AIVMD), and other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia, or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease.

Another set of embodiments provide for pharmaceutical compositions and methods, comprising a modulator and a pharmaceutically acceptable carrier or diluent, mentioned herein which can be administered to a cell, an animal, or a human subject to increase the expression or activity of FNIP1 and/or FNIP2 in the cell, animal or human subject, which can be used to treat, ameliorate, or prevent diseases, particularly inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, and cardiomyopathy; as well as cancers, such as fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas, pancreatic neuroendocrine tumors, pheochromocytomas, endolymphatic sac tumors, kidney cysts, lung cysts, and other diseases that are linked to loss-of-function of FNIP1 and/or FNIP2.

Treatment Methods

In certain embodiments, methods of treatment comprising administration of the pharmaceutical compositions to a cell, an animal or a human subject can vary in terms of composition, quantity of doses, and scheduling of doses. A unit dose is a pre-determined therapeutically effective amount of pharmaceutical composition that is administered. Unit doses can vary depending on various factors, including but not limited to, weight, age, gender, severity of symptoms, medical history, and aggressiveness of treatment. A schedule is the frequency of administration of unit doses. The size of a unit dose and the schedule of administration of the pharmaceutical composition can be determined by a person of ordinary skill in the art and are incorporated in certain embodiments herein.

In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with one or more other pharmaceutical agents. In one embodiment, the one or more other pharmaceutical agents are designed to treat a different disease, disorder, symptom, or condition compared to the one or more pharmaceutical compositions described herein. In another embodiment, the one or more other pharmaceutical agents are designed to treat the same disease, disorder, symptom, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, the one or more other pharmaceutical agents are co-administered with one or more pharmaceutical compositions described herein to produce an additive effect. In certain embodiments, the one or more other pharmaceutical agents are co-administered with one or more pharmaceutical compositions described herein to produce a synergistic or supra-additive effect, wherein the co-administration of the pharmaceutical composition and agent results in an effect that is greater than the sum of the effects of administering either pharmaceutical composition or agent alone. In another embodiment, the one or more other pharmaceutical agents are co-administered with one or more pharmaceutical compositions described herein to treat an undesired side effect of one or more pharmaceutical compositions described herein. In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with one or more other pharmaceutical agents to treat an undesired side effect of the one or more other pharmaceutical agents. In certain embodiments, one or more pharmaceutical compositions described herein are co-administered with one or more other pharmaceutical agents to prevent or delay the onset of symptoms, slow disease progression, improve the therapeutic efficacy of the one or more pharmaceutical compositions, or to otherwise improve patient outcomes.

In some embodiments, one or more pharmaceutical compositions described herein and one or more other pharmaceutical agents are prepared together in a single formulation. In other embodiments, one or more pharmaceutical compositions described herein and one or more other pharmaceutical agents are prepared separately. In certain embodiments, the one or more pharmaceutical agents are administered following administration of one or more pharmaceutical compositions described herein. In certain embodiments, the one or more pharmaceutical agents are administered prior to administration of one or more pharmaceutical composition described herein. In certain embodiments, the pharmaceutical agent is co-administered at the same time as a pharmaceutical composition described herein.

In certain embodiments, the one or more pharmaceutical composition described herein and the one or more other pharmaceutical agent are antisense modulators. In certain embodiments, the one or more pharmaceutical composition described herein is an antisense modulator, and the one or more other pharmaceutical agent is a small molecule modulator. In other embodiments, the one or more pharmaceutical composition described herein and the one or more other pharmaceutical agent can independently comprise modulators such as antisense modulators, other oligonucleotide modulators (e.g., ribozyme, deoxyribozyme, or aptamers), antibody modulators, peptide modulators, small molecule modulators, and/or nucleic acid vectors. In certain embodiments, one or more pharmaceutical agents that can be co-administered with one or more pharmaceutical compositions described herein include Riluzole (Rilutek), Dexpramipexole, Edaravone, Tofersen, Baclofen (Lioresal), or other drug that is typically administered to treat or ameliorate symptoms in ALS. In another embodiment, one or more pharmaceutical agents that can be co-administered with one or more pharmaceutical compositions described herein include one or more drugs that are typically administered to treat, ameliorate, or manage symptoms in a TDP-43 proteinopathy. In another embodiment, one or more pharmaceutical agents that can be co-administered with one or more pharmaceutical compositions described herein include one or more drugs that are typically administered to treat, ameliorate, or manage symptoms in oxidative stress, obesity, anemia or ischemic diseases. In yet another embodiment, one or more pharmaceutical agents that can be co-administered with one or more pharmaceutical compositions described herein include one or more drugs that are typically administered to treat or ameliorate symptoms in diseases such as inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, B cell deficiency, cardiomyopathy, spontaneous pneumothorax, as well as cancers. In certain embodiments, one or more pharmaceutical agents that can be co-administered with one or more pharmaceutical compositions described herein include drugs that alleviate pain, inflammation or other symptoms (e.g., COX inhibitors, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, pranoprofen, carprofen, indomethacin, folinic acid, tiaprofenic acid, diclofenac, niflumic acid, diazepines or benzodiazepines (e.g., Diazepam), barbiturate); or drugs that improve uptake or delivery, such as blood thinners (e.g., aspirin, warfarin); or antibacterial, antiviral, antibiotic; or other drug that provides at least one benefit, including treatment efficacy, symptom alleviation, drug tolerance, or side effect mediation, in a therapeutic setting.

In certain embodiments, one or more pharmaceutical agents that can be co-administered with a pharmaceutical composition to reduce or inhibit the expression or activity of FNIP1 and/or FNIP2 described herein include, but are not limited to, an additional FNIP1 and/or FNIP2 modulator, or other modulator that can reduce the levels of pathological TDP-43 aggregates in the cytoplasm, or increase the levels of functional TDP-43 in the nucleus, or achieve a combination of both. In certain embodiments, the dose of a co-administered pharmaceutical agent is lower than the dose that would be administered if the co-administered pharmaceutical agent was administered alone. In certain embodiments the dose of a co-administered pharmaceutical agent is higher than the dose that would be administered if the co-administered pharmaceutical agent was administered alone. In certain embodiments the dose of a co-administered pharmaceutical agent is the same as the dose that would be administered if the co-administered pharmaceutical agent was administered alone.

Certain Indications

In certain embodiments, provided herein are methods of treatment of a human subject diagnosed with a neuromuscular or neurodegenerative disease, such as, for example, ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), or other TDP-43 proteinopathies described herein, comprising administering one or more pharmaceutical compositions described herein to the human individual. In certain embodiments, provided herein are methods for prophylactically reducing or inhibiting FNIP1 and/or FNIP2 expression or activity in a human subject, wherein the human subject is at risk for developing a neuromuscular or neurodegenerative disease, including but not limited to, ALS, or other TDP-43 proteinopathies described herein. In some embodiments, provided herein are methods of treatment of a human subject diagnosed with oxidative stress, obesity, anemia or ischemic disease, such as, for example, chronic anemia, cardiovascular disease, myocardial ischemia or peripheral vascular disease, comprising administering one or more pharmaceutical compositions described herein to the human individual. In certain embodiments, provided herein are methods for prophylactically reducing or inhibiting FNIP1 and/or FNIP2 expression or activity in a human subject, wherein the human subject is at risk for developing oxidative stress, obesity, anemia, or ischemic diseases, such as cardiovascular disease, myocardial ischemia, or peripheral vascular disease.

In certain embodiments, provided herein are methods of treatment of a human subject diagnosed with a disease, such as von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers, such as fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas, pancreatic neuroendocrine tumors, pheochromocytomas, endolymphatic sac tumors, kidney cysts, lung cysts, or other cancers described herein, comprising administering one or more pharmaceutical compositions described herein to the human individual. In certain embodiments, provided herein are methods for prophylactically increasing FNIP1 and/or FNIP2 expression or activity in a human subject, wherein the human subject is at risk for developing von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers described herein. In some embodiments, provided herein are methods of treatment of a human subject diagnosed with inflammatory diseases, such as, for example, allergy, asthma, atherosclerosis, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), psoriasis, renal fibrosis, reperfusion injury, rheumatoid arthritis, transplant rejection, tubular ischemia-reperfusion damage, vascular inflammation, or neuroinflammatory diseases described herein, comprising administering one or more pharmaceutical compositions described herein to the human individual. In certain embodiments, provided herein are methods for prophylactically increasing FNIP1 and/or FNIP2 expression or activity in a human subject, wherein the human subject is at risk for developing an inflammatory disease described herein.

In certain embodiments, provided herein are methods of treatment of a human subject in need thereof by administering to the human individual a therapeutically effective amount of an antisense modulator targeting one or more FNIP1 and/or FNIP2 nucleic acids disclosed by SEQ ID NOs: 1-15 herein. In one embodiment, administration of a therapeutically effective amount of an antisense modulator targeted to a FNIP1 and/or FNIP2 nucleic acid disclosed by SEQ ID NOs: 1-15 herein is accompanied by monitoring of FNIP1 and/or FNIP2 levels in the human individual, to determine the individual's response to administration of the antisense modulator. In some embodiments, provided herein are methods of treatment of a human subject in need thereof by gene therapy, comprising administering to the human individual a therapeutically effective amount of a nucleic acid vector described herein. In one embodiment, administration of a therapeutically effective amount of a nucleic acid vector is accompanied by monitoring of FNIP1 and/or FNIP2 levels in the human individual, to determine the individual's response to administration of the nucleic acid vector modulator. In certain embodiments, provided herein are methods of treatment of a human subject in need thereof by administering to the human individual a therapeutically effective amount of a modulator described herein, such as, for example, other oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, that targets the expression or activity of FNIP1 and/or FNIP2. In one embodiment, administration of a therapeutically effective amount of a modulator described herein, such as, for example, other oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, is accompanied by monitoring of FNIP1 and/or FNIP2 levels in the human individual to determine the individual's response to administration of the modulator. A human subject's response to administration of the antisense or other modulator can be used by a physician to determine the dose, schedule, and duration of therapeutic intervention.

In certain embodiments, administration of an antisense modulator targeted to a FNIP1 and/or FNIP2 nucleic acid disclosed by SEQ ID NOs: 1-15 herein, results in a decrease of FNIP1 and/or FNIP2 mRNA or protein expression by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99%, or a range defined by any two of these values. In other embodiments, administration of an antisense modulator targeted to a FNIP1 and/or FNIP2 nucleic acid disclosed by SEQ ID NOs: 1-15 herein, results in an increase of FNIP1 and/or FNIP2 mRNA or protein levels by at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1900, 2900, 3900, 4900, 5900, 6900, 7900, 8900, or 9900%, or a range defined by any two of these values. In certain embodiments, administration of an antisense modulator targeted to a FNIP1 and/or FNIP2 nucleic acid disclosed by SEQ ID NOs: 1-15 herein, results in improved motor function and respiration in a human subject. In certain embodiments, administration of a FNIP1 and/or FNIP2 antisense modulator improves motor function and respiration by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99%, or a range defined by any two of these values. In certain embodiments, administration of a modulator, such as a nucleic acid vector, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, results in a decrease of FNIP1 and/or FNIP2 mRNA or protein expression by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99%, or a range defined by any two of these values. In certain embodiments, administration of a modulator, such as a nucleic acid vector, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, results in an increase of FNIP1 and/or FNIP2 mRNA or protein levels by at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1900, 2900, 3900, 4900, 5900, 6900, 7900, 8900, or 9900%, or a range defined by any two of these values. In certain embodiments, administration of a modulator, such as a nucleic acid vector, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, results in improved motor function and respiration in a human subject. In certain embodiments, administration of a modulator, such as a nucleic acid vector, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, improves motor function and respiration by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising an antisense modulator targeted to FNIP1 and/or FNIP2, wherein the modulator is capable of decreasing the expression or activity of FNIP1 and/or FNIP2, are used for the preparation of a medicament for treating a patient diagnosed with or susceptible to a disease, particularly neuromuscular or neurodegenerative disease, such as, for example ALS, FTLD, or other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases described herein. In other embodiments, pharmaceutical compositions comprising a modulator described herein, such as a nucleic acid vector, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, wherein the modulator is capable of decreasing the expression or activity of FNIP1 and/or FNIP2, are used for the preparation of a medicament for treating a patient diagnosed with or susceptible to a disease, particularly neuromuscular or neurodegenerative disease, such as, for example ALS, FTLD, or other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases described herein.

In other embodiments, pharmaceutical compositions comprising an antisense modulator targeted to FNIP1 and/or FNIP2, wherein the modulator is capable of increasing the expression or activity of FNIP1 and/or FNIP2, are used for the preparation of a medicament for treating a patient diagnosed with or susceptible to a disease, particularly inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers described herein. In other embodiments, pharmaceutical compositions comprising a modulator described herein, such as a nucleic acid vector, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, wherein the modulator is capable of increasing the expression or activity of FNIP1 and/or FNIP2, are used for the preparation of a medicament for treating a patient diagnosed with or susceptible to a disease, particularly inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers described herein.

Methods of Development

In certain embodiments, provided herein are methods of development of a modulator, such as an antisense modulator, oligonucleotide modulator, antibody modulator, peptide modulator, or small molecule modulator, which are capable of modulating the expression or activity of FNIP1 and/or FNIP2. In certain embodiments, the methods of development of a modulator are entirely computational. In some embodiments, the methods of development of a modulator involve biochemical methods, such as screens and selections. In some embodiments, the methods of development of a modulator involve a combination of biochemical and computational methods. Such methods of development involve standard practices that are known to a person who is skilled in the art and are incorporated in certain embodiments herein.

In some embodiments, computational methods can involve artificial intelligence or machine learning software, rely on large molecular databases, utilize high throughput analysis, or any combination thereof. In one embodiment, a computational method can be used to screen existing drug candidates, which can be repurposed for use as modulators herein. Certain methods for computational drug repurposing are described by Li et al. (Li et al., Briefings in Bioinformatics, 17(1): 2-12 (2016)) and Hodos et al. (Hodos et al., Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 8(3): 186-210 (2016)), which, along with the references cited therein, are incorporated by reference herein in their entirety. Other computational methods are well known to a person of ordinary skill in the art and are included in various embodiments herein.

Antisense Modulator

In certain embodiments, provided herein are methods of development of antisense modulators that are capable of targeting one or more transcripts of FNIP1 and/or FNIP2 described by SEQ ID NOs: 1-15, or its associated genes or pathways, thereby modulating the expression or activity of FNIP1 and/or FNIP2. Antisense modulators include compounds that do not act through the RNAi pathway, such as antisense oligonucleotides, as well as compounds that act through the RNAi pathway, such as siRNA, shRNA, or miRNA. The design of an appropriate antisense modulator is critical to its safety and effectiveness as a therapy.

Several guiding principles can be used individually or in conjunction with one another to design antisense oligonucleotide modulators. Firstly, the sequence of the modulator should be antisense to the target genetic sequence of choice. However, mismatches or imperfect complementarity between the antisense oligonucleotide modulator and target sequence can be tolerated as described elsewhere herein. Secondly, the sequence of the antisense oligonucleotide modulator is ideally not more than 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 nucleotides in length. In a preferred embodiment, the antisense oligonucleotide modulator is between 12 to 30 nucleotides in length. Thirdly, the antisense oligonucleotide modulator ideally targets a region of the target nucleic acid that is accessible and does not contain stable secondary structures. Fourthly, the antisense oligonucleotide modulator should possess sufficient binding energy to the target nucleic acid molecule. The antisense oligonucleotide modulator can be modified to improve stability, delivery and efficacy, as described elsewhere herein. Methods and procedures for designing and developing antisense oligonucleotides are well known in the art, for example, as described by Chan et al. (Chan et al., Clinical and Experimental Pharmacology and Physiology, 33:533-540 (2006)), which along with references cited therein, is incorporated by reference in its entirety. Delivery systems for antisense oligonucleotides are described herein and further described by Chan et al. and Zhao et al. (Zhao et al., Expert Opinion on Drug Delivery, 6(7): 673-686 (2009)), both of which along with references cited therein, are incorporated by reference in their entirety and are known to a person of ordinary skill in the art.

The methods detailed below are relevant to the screening and development of siRNA, shRNA, or miRNA, and any procedures detailed below for the screening of siRNA are also applicable to shRNA and miRNA. The methods detailed below are also relevant to the screening and development of antisense oligonucleotides, and any procedures detailed below for the screening of siRNA are also applicable to antisense oligonucleotide modulators.

The design of an appropriate siRNA sequence is critical to the performance of RNAi therapies. siRNA sequences are chosen based on several guiding principles that can be used individually or in conjunction with one another. Firstly, the siRNA should be antisense to the target genetic sequence of choice. Secondly, the siRNA sequence can ideally be chosen to be in the range of 19 to 29 nucleotides in length. Thirdly, the target area of the genetic sequence should be at least 100 nucleotides from the initiation codon and 50 nucleotides away from the stop codon. Fourthly, the introduction of 3′-d(TT) overhangs are recommended. Fifthly, structural considerations should be made to promote stability and effectiveness of the siRNA (e.g., the GC ratio limited to between 45% and 55%, or the elimination of poly-C or poly-G sequences). Certain tools (e.g., http://sima.wi.mit.edu/) can be used to assist in the development of appropriate siRNA designs. A BLAST search is recommended to be performed to eliminate siRNA candidates with low specificity to the targeted genetic sequence. In certain cases, the siRNA sequence or multiple siRNA sequences can be integrated into a strand of double-stranded RNA. The siRNA modulator can be modified to improve stability, delivery and efficacy, as described elsewhere herein. Methods and procedures for developing oligonucleotides for RNAi treatment are further described by Duxbury and Whang (Duxbury and Whang, Journal of Surgical Research, 117:339-344 (2004)), which along with references cited therein, are incorporated by reference in its entirety and are known to a person of ordinary skill in the art. Delivery systems for RNAi treatments are further described by Deng et al. (Deng et al., Gene, 538(2):217-227 (2014)), which along with references cited therein, is incorporated by reference in its entirety and are known to a person of ordinary skill in the art.

Small Molecule

In certain embodiments, provided herein are methods of development of small molecule modulators that are capable of targeting FNIP1 and/or FNIP2, or its associated genes or pathways, thereby modulating the expression or activity of FNIP1 and/or FNIP2. Provided herein are methods of development of small molecule modulators that target nucleic acids encoding FNIP1 and/or FNIP2. Also provided herein are methods of development of small molecule modulators that target FNIP1 and/or FNIP2 proteins.

In some embodiments, methods of development of small molecule modulators that target nucleic acids encoding FNIP1 and/or FNIP2 include computational methods. Computational methods described by Manigrasso et al. (Manigrasso et al., Chem, 7(11): 2965-2988 (2021)) are representative of some of the computational methods available, and this reference along with references cited therein, are incorporated by reference herein. In one embodiment, the 2D and/or 3D structure of a target region of a nucleic acid encoding FNIP1 and/or FNIP2 is computationally predicted. In one embodiment, the 2D and/or 3D structure of a target region of a nucleic acid encoding FNIP1 and/or FNIP2 is determined using one or more experimental methods that are well known in the art, including but not limited to, NMR, SHAPE, X-ray crystallography, etc. In one embodiment, a computational library of small molecule modulators is computationally docked individually with a target region of a nucleic acid encoding FNIP1 and/or FNIP2. In one embodiment, a computational method is used to determine the binding energy of each small molecule modulator to a target region of a nucleic acid encoding FNIP1 and/or FNIP2. In yet another embodiment, new small molecule modulators are created for computational screening from an amalgamation of existing molecules or atoms from one or more databases. In certain embodiments, small molecule modulators that are predicted to have favorable binding energies to a target region of a nucleic acid encoding FNIP1 and/or FNIP2 are prioritized for further analysis and development.

In some embodiments, methods of development of small molecule modulators that target FNIP1 and/or FNIP2 proteins include computational methods. Computational methods described by Mendez-Lucio et al. (Mendez-Lucio et al., Nature Communications, 11, 10 (2020)), Dallakyan and Olson (Dallakyan and Olson, Hempel et al. (ed.) Chemical Biology: Methods and Protocols, Chapter 19, Methods in Molecular Biology pg 243-250 (2015)), Zoete et al. (Zoete et al., Journal of Chemical Information and Modeling, 56: 1399-1404 (2016)), and Merk et al. (Merk et al., Molecular Informatics, 37: 1700153 (2018)) are representative of some of the computational methods available, and these reference along with references cited therein, are incorporated by reference herein. In one embodiment, a computational library of small molecule modulators is computationally docked individually with a target protein, such as FNIP1 and/or FNIP2. In one embodiment, a computational method is used to determine the binding energy of each small molecule modulator to a target protein, such as FNIP1 and/or FNIP2. In yet another embodiment, new small molecule modulators are created for computational screening from an amalgamation of existing molecules or atoms from one or more databases. In certain embodiments, small molecule modulators that are predicted to have favorable binding energies to the target protein are prioritized for further analysis and development.

In certain embodiments, methods of development of small molecule modulators include high throughput biochemical screening methods, which are known to a person of ordinary skill in the art. Physical libraries of small molecules are built or obtained from commercially available sources. These libraries are screened against a target molecule of choice, such as a nucleic acid encoding FNIP1 and/or FNIP2, or FNIP1 and/or FNIP2 protein, by introducing the small molecules to the target molecule of choice and then implementing a washing or separating method to determine binding affinity and/or specificity. A biochemical or cell-based assay or a series of biochemical or cell-based assays can be used to determine structural and chemical properties of the small molecules or molecular complexes that are formed between the small molecule and the target molecule of choice. Controls or negative selection steps can be used to screen out small molecules with off-target binding activity to other molecules that are not the target molecule of choice. Further, screening at different concentrations of the small molecule against the target molecule of choice is used to determine other properties such as IC50, EC50, potency, and the like. Further description of methods and procedures for developing small molecule modulators via high-throughput screening are described by Cronk (Cronk, Drug Discovery and Development (Second Edition) Chapter 8, pp. 95-117 (2013)), which along with references cited therein, are incorporated by reference in its entirety and are known to a person of ordinary skill in the art. Other methods are well known to a person of ordinary skill in the art and are included in various embodiments herein.

In certain embodiments, methods of development of small molecule modulators include fragment-based discovery techniques that are known to a person of ordinary skill in the art. These methods involve screening of a library of small molecular fragments that contain one or more binding epitopes for binding affinity and/or specificity to a target molecule, such as a nucleic acid encoding FNIP1 and/or FNIP2, or FNIP1 and/or FNIP2 protein. Typically, the small molecular fragments have a molecular mass of around 120-250 daltons. In certain cases, these fragment-based discovery methods are combined with computational methods, some of which are described below. Examples of fragment-based discovery techniques include lead identification by fragment evolution, lead identification by fragment linking, lead identification by fragment self-assembly, and lead progression by fragment optimization, which are described herein. Often, assessment of target molecule binding sites, the development of fragment complexes, and subsequent determination of efficacy or specificity of binding is informed by structural, morphological, and chemical data acquired from assessment tools such as nuclear magnetic resonance spectroscopy, mass spectrometry, or X-ray crystallography.

In lead identification by fragment evolution, a library of fragments is applied to the target molecule of choice, and the strength and specificity of binding is determined. The fragments with higher binding specificity are then reacted or evolved with various other fragments to form fragment complexes that are screened for having even higher binding specificity.

In lead identification by fragment linking, fragment libraries are screened through multiple binding sites of a target molecule of choice for binding specificity. Two or more fragments with high binding specificity to two or more nearby binding sites on a target molecule of choice are chemically linked together.

In lead identification by fragment self-assembly, also termed combinatorial chemistry, a library of fragments capable of self-assembly is introduced to a target molecule of choice. The fragments are allowed to bind to the target molecule of choice in a manner that produces a complex that inhibits the expression or activity of the target molecule of choice. The various fragments are capable of assembling together while bound to the target molecule of choice via complementary reactive groups. Once assembled, these fragment complexes can then be isolated to assess their chemical and structural properties.

In lead progression by fragment optimization, a library of fragments is used to modify the properties of an existing modulator or fragment complex. Typically, this method is used to address the optimization of certain properties, such as selectivity, solubility, stability, or efficacy.

Examples and further discussion of methods and procedures for the development of small molecule modulators via fragment-based discovery are described by Rees et al. (Rees et al., Nature Reviews Drug Discovery, 3(8): 660 (2004)), Erlanson et al. (Erlanson et al., Journal of Medicinal Chemistry, 47(14): 3463-3482 (2004)), and Congreve et al. (Congreve et al., Journal of Medicinal Chemistry, 51(13): 3661-3680 (2008)), all of which along with references cited therein, are incorporated by reference in its entirety and are known to a person of ordinary skill in the art. Other methods are well known to a person of ordinary skill in the art and are included in various embodiments herein.

Antibody and Related Protein

In certain embodiments, provided herein are methods of development of antibody and other related protein modulators that are capable of targeting FNIP1 and/or FNIP2, or its associated genes or pathways, thereby modulating the expression or activity of FNIP1 and/or FNIP2. In some embodiments, computational methods are used to develop antibody and related protein modulators. Computational methods as described by Chevalier et al. (Chevalier et al., Nature, 550, 7674 (2017)) are representative of some of the computational methods available, and this reference along with references cited therein, are incorporated by reference herein. In other embodiments, biochemical methods are used to develop, screen and produce antibody or related protein modulators. Such methods use a common set of methods that are known to a person of ordinary skill in the art. Throughout this specification, the antibodies referred to include all forms of antibodies, including but not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and monobodies.

In certain embodiments, antibodies are developed by introducing an antigen that activates an immunological response in an animal species, such as, but not limited to, rabbit, mouse, rat, hamster, guinea pig, goat, sheep, chicken, or humans, and harvesting the resulting antibodies from blood or, in some cases, eggs, tissue, or other fluids. In certain embodiments, an adjuvant is also used alongside the antigen to increase the level of immunological response. Examples of commonly used adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum salts, Quil A, Iscoms, Montanide, TiterMax, and RIBI.

Methods of development or production of polyclonal antibodies are known to persons skilled in the art. One such process can include either single or multiple introductions of at least one antigen or antigen/adjuvant mixture into the animal species of choice, clinical monitoring of antibody levels, and antibody collection once sufficient antibody levels are reached.

Methods of development or production of monoclonal antibodies are known to persons skilled in the art. In one common embodiment, BALB/c mice are used as the animal species for antigen injection, although other species mentioned above can be used. One such process can include either single or multiple introductions of at least one antigen or antigen/adjuvant mixture into the animal species of choice and clinical monitoring of antibody levels. A final injection of just antigen with no adjuvant is typically administered prior to harvesting B cells from the animal. The B cells are fused with non-secreting myeloma cells to form hybridoma B cells, which are cloned and selected for antigen specificity to a target molecule using one of any number of screening methods known to persons skilled in the art. Typical production processes utilize antigen-specific hybridoma B cells to generate identical copies of the antibody. Other methods, such as in vitro display selection (see below), can be used for the development of monoclonal antibodies and are known to a person of ordinary skill in the art.

In certain embodiments, provided herein are methods to further increase production levels of monoclonal antibodies. In the ascites method, the chosen monoclonal antibody-producing hybridoma cells are isolated and injected into an animal species, such as mice, which instigates the growth of ascites in areas, such as the abdominal cavity. In certain cases, a priming agent, such as pristine, is first injected into the animal to suppress immune response, promote the secretion of serous fluid, and slow hybridoma cell clearance. After incubation for several days to weeks, antibodies can be extracted from the animal directly from the ascites. In in vitro methods for increasing production levels of monoclonal antibodies, the chosen monoclonal antibody-producing hybridoma cells are isolated and cultured, often in a nutrient medium and/or serum. Single-compartment culture systems, such as culture flasks, roller bottles, or gas-permeable bags can be used for smaller scale production. In larger scale production, double-compartment culture systems, such as hollow-fiber systems, fermenters, perfusion-tank systems, airlift reactors, or continuous-culture systems can be used.

In certain embodiments, provided herein are methods for the development or production of chimeric or humanized antibodies, which are known in the art. The development or production of chimeric or humanized antibodies can be accomplished through genetic engineering of an animal genome to contain human or human-like coding segments in the antibody. Further modification of antibodies can also be effected by genetic engineering of the genome of B cells, hybridoma cells, or vectors.

A 1999 report by the National Research Council (Monoclonal Antibody Production: A Report of the Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council (1999)) and an article by Leenars and Hendriksen (Leenars and Hendriksen, Ilar Journal, 46(3): 269-279 (2005)) detail further certain procedures for synthesizing antibodies, both of which along with references cited therein, are incorporated by reference in their entirety and are known to a person of ordinary skill in the art. Cell-free antibody synthesis procedures are described by Stech and Kubick (Stech and Kubick, Antibodies, 4: 12-33 (2015)), which along with references cited therein, is incorporated by reference in its entirety and are known to a person of ordinary skill in the art.

In one embodiment, an antibody modulator is screened for and developed using an antibody development, screening, or production method, or any combination of methods thereof detailed above.

Aptamer

In certain embodiments, provided herein are methods of development of aptamer modulators, including oligonucleotide, oligopeptide, or polypeptide aptamers, which are capable of targeting FNIP1 and/or FNIP2, or its associated genes or pathways, thereby modulating the expression or activity of FNIP1 and/or FNIP2. In certain embodiments, in vitro selection, also referred to as SELEX or in vitro evolution, can be used to develop and screen for aptamer modulators, such as oligonucleotide aptamers, which have strong binding affinities to a target molecule of choice, such as FNIP1 and/or FNIP2. In vitro selection methods are known to a person of ordinary skill in the art, and are incorporated in certain embodiments herein. In the first step, a large population of degenerate oligonucleotides is synthesized and amplified to create an oligonucleotide library using polymerase chain reaction. In the second step, the oligonucleotide library is treated with a change in temperature to renature the oligonucleotide library into stable secondary or tertiary structures. In the third step, the oligonucleotide library is introduced to the target molecule of choice, which has been immobilized to a substrate. In the fourth step, the oligonucleotides that are bound to the target molecule of choice are isolated from the rest of the oligonucleotide library via a washing method or other related method. In the fifth step, the oligonucleotides that are bound to the target molecule of choice are eluted from the target molecule of choice, typically using an oligonucleotide denaturing method such as high temperature or application of denaturing solutions, and isolated. In the sixth step, the oligonucleotides are further amplified by polymerase chain reaction and converted into the desired oligonucleotide format (e.g., single-stranded DNA, single-stranded RNA, or other oligonucleotide format). In the seventh step, the second step through the sixth step are repeatedly cycled until the desired specificity and/or binding affinity of the oligonucleotides to the target molecule of choice is reached. In certain instances, a negative selection step can be implemented between repeated cycles during which other target molecules can be used to bind to and remove oligonucleotides with specificity to those other target molecules. Other methods are well known to a person of ordinary skill in the art and are included in various embodiments.

In certain embodiments, provided herein are methods of in vitro display to develop and screen for oligopeptide and polypeptide aptamers that have strong binding affinities to a target molecule of choice, such as FNIP1 and/or FNIP2. In vitro display methods are known to a person of ordinary skill in the art and include, but are not limited to, phage display, cell surface display, ribosome display, mRNA display, DNA display, and in vitro compartmentalization.

Phage display can be used as a selection process that identifies and screens the binding affinity and specificity of a collection of oligopeptide or polypeptide aptamers to a target molecule of choice, using common strains of bacteriophage (e.g., M13, fd, or f1) as a means of surface peptide display. In the first step, a combinatorial library of oligopeptide or polypeptide fragments is identified, and a nucleotide library is built encoding the combinatorial library of peptide fragments. In the second step, the nucleotide sequences from the nucleotide library are coupled to nucleotide sequences encoding the major or minor coat proteins in the genome of the bacteriophage of choice. In the third step, the bacteriophages are introduced into a bacteria host (e.g., E. coli) to be replicated. The bacteriophages that are replicated express the various peptide fragments from the combinatorial library on the bacteriophage surface and contain the corresponding modified genome. In the fourth step, the bacteriophages are introduced to the target molecules of choice that have been immobilized on a substrate. In the fifth step, the bacteriophages that are bound to the target molecule of choice are isolated from the rest of the bacteriophages via a washing method or other related method. In the sixth step, the remaining, bound bacteriophages are eluted via a method such as increased temperature or a denaturing solution. In the seventh step, the eluted bacteriophages are cycled through the third through the sixth step until the desired level of binding affinity and/or specificity towards the target molecule of choice is achieved. In the eighth step, the genetic sequence of the polypeptide or oligopeptide aptamer with the desired level of specificity and/or binding affinity towards the target molecule of choice is isolated from the bacteriophage and can be used to produce additional copies of the polypeptide or oligopeptide aptamer. In certain instances, a negative selection step can be implemented between repeated cycles during which other target molecules can be used to bind to and remove polypeptide or oligopeptide aptamers with specificity to those other target molecules.

Cell surface display can be used as a selection process that identifies and screens the binding affinity and specificity of a collection of oligopeptide or polypeptide aptamers to a target molecule of choice using a cell or collection of cells (e.g., bacteria or yeast) as a means of surface peptide display. The selection process of aptamers using cell surface display is similar to that of phage display. In the first step, a combinatorial library of peptide fragments is identified, and the nucleotide library is built encoding the combinatorial library of peptide fragments. In the second step, the nucleotide sequences from the nucleotide library are coupled to nucleotide sequences encoding an outer membrane protein and introduced into the cell of choice, either through transformation, introduction via a vector, or other similar method known in the art. In the third step, the cell is allowed to replicate as well as to transcribe and translate the nucleotide sequence encoding the peptide fragment linked to an outer membrane protein such that the peptide fragment is displayed on the cell surface. In the fourth step, the cells are introduced to the target molecules of choice that have been immobilized on a substrate. In the fifth step, the cells that are bound to the target molecule of choice are isolated from the rest of the cells via a washing method or other related method. In the sixth step, the remaining, bound cells are eluted via a method such as increased temperature or a denaturing solution. In the seventh step, the eluted cells are cycled through the third through the sixth step until the desired level of specificity and/or binding affinity towards the target molecule of choice is achieved. In the eighth step, the genetic sequence of the polypeptide or oligopeptide aptamer with the desired level of specificity towards the target molecule of choice is isolated from the cell, which can be used to produce additional copies of the aptamer. In certain instances, a negative selection step can be implemented between repeated cycles during which other target molecules can be used to bind to and remove polypeptide or oligopeptide aptamers with specificity to those other target molecules.

Ribosome display can be used as a selection process that identifies and screens for polypeptide or oligopeptide aptamers that bind with high affinity and/or specificity to a target molecule of choice, such as FNIP1 and/or FNIP2. The ribosome display process involves a library of mRNA molecules that code for the library of aptamers identified for screening. These mRNA molecules are modified to have a ribosome binding site at the 5′ end and a spacer sequence with no stop codon on the 3′ end. In the first step, the modified mRNA molecules are prepared. In the second step, the modified mRNA molecules are translated in vitro, which causes an mRNA-peptide-ribosome complex to be formed. In the third step, the mRNA-peptide-ribosome complex is isolated and introduced to the target molecules of choice that have been immobilized on a substrate. In the fourth step, the complexes that bind to the target molecules of choice are sorted and isolated. In the fifth step, the isolated complexes bound to the target molecules of choice are eluted and dissociated, and the mRNA molecules that correspond to those complexes with preferential binding to the target molecules of choice are recovered. In the sixth step, those mRNA molecules are reverse transcribed and amplified via polymerase chain reaction to produce DNA sequences encoding aptamers with high specificity and/or binding affinity to the target molecules of choice. In the seventh step, the first step through the sixth step are repeated using the nucleotides obtained from the previous sixth step until the desired level of specificity and/or affinity towards the target molecules of choice is obtained. The genetic sequence of the aptamer can then be used to produce additional copies of the aptamer. In certain instances, a negative selection step can be implemented between repeated cycles during which other target molecules can be used to bind to and remove polypeptide or oligopeptide aptamers with specificity to those other target molecules.

mRNA display can be used as a selection process that identifies and screens for polypeptide or oligopeptide aptamers that bind with high affinity and/or specificity to a target molecule of choice, such as FNIP1 and/or FNIP2. The mRNA display process involves creating a library of mRNA molecules that code for the library of aptamers identified for screening. These mRNA molecules are modified to have a short DNA linker with puromycin at the 3′ end. In the first step, these modified mRNA molecules are prepared. In the second step, the modified mRNA molecules are translated in vitro, which causes an mRNA-peptide complex to be formed from the reaction between puromycin and the nascent polypeptide. In the third step, the mRNA-peptide complex is isolated and introduced to the target molecules of choice that have been immobilized on a substrate. In the fourth step, the complexes that bind to the target molecules of choice are sorted and isolated. In the fifth step, the isolated complexes bound to the target molecules of choice are eluted and dissociated, and the mRNA molecules that encode for oligopeptide or polypeptide aptamers with high affinity and/or specificity to the target molecules of choice are recovered. In the sixth step, those mRNA molecules are reverse transcribed and amplified via polymerase chain reaction to form DNA sequence encoding the aptamers. In the seventh step, the first step through the sixth step are repeated using the nucleotides obtained from the previous sixth step until the desired level of specificity and/or affinity towards the target molecules of choice is obtained. The genetic sequence of the aptamer can then be used to produce additional copies of the aptamer. In certain instances, a negative selection step can be implemented between repeated cycles during which other target molecules can be used to bind to and remove polypeptide or oligopeptide aptamers with specificity to those other target molecules.

DNA display can be used as a selection process that identifies and screens for polypeptide or oligopeptide aptamers that bind with high affinity and/or specificity to a target molecule of choice, such as FNIP1 and/or FNIP2. The DNA display process involves a library of DNA sequences that code for the library of aptamers identified for screening linked to DNA sequence encoding the protein streptavidin, which forms the conjugate. These DNA sequences are further labeled with biotin. In the first step, the modified DNA sequences are prepared, and mRNA molecules are transcribed from these DNA sequences in separate compartments containing on average one member of the DNA library. In the second step, the mRNA molecules are translated in vitro, which produces a polypeptide or oligopeptide aptamer linked to streptavidin, which binds to the biotin-labeled DNA. In the third step, the DNA-peptide complex is isolated and introduced to the target molecules of choice that have been immobilized on a substrate. In the fourth step, the complexes that bind to the target molecules of choice are sorted and isolated. In the fifth step, the isolated complexes bound to the target molecules of choice are eluted and dissociated, and the DNA molecules that encode the aptamers with preferential binding to the target molecules of choice are recovered. In the sixth step, those DNA molecules are amplified via polymerase chain reaction to obtain the genetic sequence of aptamers that possess high binding affinity and/or specificity to the target molecules of choice. In the seventh step, the first step through the sixth step are repeated using the nucleotides obtained from the previous sixth step until the desired level of specificity and/or affinity towards the target molecules of choice is obtained. The genetic sequence of the aptamer can then be used to produce additional copies of the aptamer. In certain variations of this method, a different pair of molecules with high binding affinity to each other, besides streptavidin and biotin, can be used for both attachment to the DNA sequence and its corresponding conjugate that is translated from the DNA sequence that is linked to the aptamer. These are able to create similar DNA-peptide complexes described above. In other variations, puromycin or other similar protein can be attached to the DNA sequence. The puromycin can bind directly to the nascent protein without the need for a conjugate polypeptide strand to form a DNA-peptide complex, as described by Chen et al. (Chen et al., RSC Advances 3, 16251 (2013)). In certain instances, a negative selection step can be implemented between repeated cycles during which other target molecules can be used to bind to and remove polypeptide or oligopeptide aptamers with specificity to those other target molecules. Other methods are well known to a person of ordinary skill in the art and are included in various embodiments herein.

Gene Therapy

In certain embodiments, provided herein are methods of development of molecules for gene therapy to modulate the expression or activity of FNIP1 and/or FNIP2. Methods of development of molecules for gene therapy are known in the art and are included in various embodiments herein. In the case of developing gene therapy where the goal is gene augmentation, a nucleotide sequence encoding one or more functional copies of the gene of interest, such as FNIP1 and/or FNIP2, can be inserted into a nucleic acid vector. In one embodiment, functional copies of the gene are placed under control of appropriate regulatory elements, such as a cell or tissue-specific promoter, wherein the gene is expressed at appropriate levels and/or at appropriate times and/or in appropriate cells or tissues to exert a beneficial or therapeutic effect for a disease. In one embodiment, the promoter is a CNS-specific promoter. In another embodiment, a modulator that can increase the activity or expression of the gene of interest is inserted into a nucleic acid vector. In another embodiment, the nucleic acid vector carrying the gene of interest or other modulator is designed to target a specific tissue, such as by selecting an appropriate viral vector that is specific to a particular tissue, or by any other means known in the art. In the case of developing gene therapy where the goal is gene suppression, a nucleotide sequence encoding at least one modulator that can suppress the expression, or the activity of the gene of interest, such as FNIP1 and/or FNIP2, is inserted into a nucleic acid vector. Methods of development of gene therapy for clinical applications are described in Sung et al. (Sung et al., Biomaterials Research, 23, Article number: 8 (2019)) and Kumar et al. (Kumar et al., Mol. Ther. Methods Clin. Dev., 3, 16034 (2016)), which together with the references cited therein, are incorporated herein in their entirety.

In the case of developing gene therapy where the goal is gene editing, the development method depends on the particular endonuclease system and desired effects on the cell. A key consideration is ensuring the specificity of targeting of the endonuclease to a specific DNA or RNA sequence, in order to reduce side effects from mis-targeting of the endonuclease. In the case of Transcription activator-like effector nucleases (TALEs/TALENs), DNA specificity is determined by a DNA Binding Domain (DBD) containing on average 1.5-33.5 tandem repeats of 34 amino acid sequences (termed monomers), wherein each monomer recognizes a specific nucleotide. Although the sequence of each monomer is highly conserved, they differ primarily in two positions (the 12th and 13th) named as repeat variable di-residues (RVDs). Recent reports have found that the identity of these two residues determines the nucleotide binding specificity of each TALE repeat in a simple cipher that specifies the target base of each RVD (NI=A, HD=C, NG=T, NN=G). Thus, each monomer targets one nucleotide, and the linear sequence of monomers in a TALE specifies the target DNA sequence in the 5′ to 3′ direction. In one embodiment, a computational method is employed to design an appropriate sequence of monomers in a TALE to target a specific DNA sequence. Methods for developing TALE/TALENs are described in Zhang et al. (Zhang et al., Molecular Therapy: Methods & Clinical Development, 13, 310-320 (2019)), which along with references cited therein, is incorporated by reference herein in their entirety. Although less modular than TALENs, Zinc-finger nucleases (ZFN) can be designed to target a specific DNA sequence through a combination of certain design rules coupled with in vitro selection techniques. Such development and screening methods for ZFNs are described in detail in Chandrasegaran and Carroll (Chandrasegaran and Carroll, Journal of Molecular Biology, 428(5), 963-989 (2016)), which along with the references cited therein, are incorporated by reference in its entirety herein.

In one embodiment of the CRISPR system, the specificity of targeting is determined by a gRNA, which directs the endonuclease to bind to a specific DNA or RNA sequence that is complementary to the gRNA. Several studies have established key rules for the design of gRNA to increase the specificity of targeting a nucleotide sequence. For example, in the case of the Cas9 nuclease, a canonical protospacer adjacent motif (PAM) site comprising the nucleotides NGG, where N is any nucleobase, must be present immediately 3′ to the sequence that is targeted by the gRNA. In the case of the Cpf1 (Cas12a) nuclease, the PAM comprises “TTTN” or “YTN”. In addition, key rules for optimizing gRNA design include avoiding poly-T sequences, limiting the GC content and avoiding a G immediately upstream of the PAM (i.e., a GNGG motif). Furthermore, it is important to check for potential off-target sites that are similar in sequence to the target site. Several computational tools have been developed for the design and/or screening of specific gRNAs and the prediction of off-target sites. Such development and screening methods for CRISPR-Cas9 are described in Wilson et al. (Wilson et al., Frontiers in Pharmacology, 9, 749 (2018)), which along with the references cited therein, are incorporated by reference herein in its entirety.

Diagnostics and Testing

In some embodiments, measuring and detecting an increase in expression levels of FNIP1 and/or FNIP2, or measuring and detecting an increase in activity of FNIP1 and/or FNIP2, can be used to determine an increased risk for or increased susceptibility to ALS. In certain embodiments, measuring and detecting an increase in signaling through FNIP1 and/or FNIP2 can be used to determine an increased risk for or increased susceptibility to ALS. In certain embodiments, measuring and detecting an increase in signaling through a pathway associated with FNIP1 and/or FNIP2, can be used to determine an increased risk for, or increased susceptibility to ALS. In some embodiments, measuring and detecting a decrease in expression levels of FNIP1 and/or FNIP2, or measuring and detecting a decrease in activity of FNIP1 and/or FNIP2, can be used to determine a decreased risk for, or decreased susceptibility to ALS. In certain embodiments, measuring and detecting a decrease in signaling through FNIP1 and/or FNIP2 can be used to determine a decreased risk for, or decreased susceptibility to ALS. In certain embodiments, measuring and detecting a decrease in signaling through a pathway associated with FNIP1 and/or FNIP2, can be used to determine a decreased risk for, or decreased susceptibility to ALS.

In certain embodiments, measuring and detecting an increase in expression or activity of FNIP1 and/or FNIP2, or an increase in signaling through a pathway associated with FNIP1 and/or FNIP2, can be used to determine an increased risk for or increased susceptibility to neuromuscular or neurodegenerative diseases, such as ALS, FTLD, Alzheimer's disease, retinal degeneration diseases such as age-related macular degeneration (AMD), or other TDP-43 proteinopathies; as well as oxidative stress, obesity, anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia and peripheral vascular disease. In other embodiments, measuring and detecting a decrease in expression or activity of FNIP1 and/or FNIP2, or a decrease in signaling through a pathway associated with FNIP1 and/or FNIP2, can be used to determine an increased risk for or increased susceptibility to inflammatory diseases, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, as well as cancers such as fibrofolliculomas, kidney tumors, clear cell renal cell carcinoma, multilocular clear cell renal carcinoma, chromophobe renal cell carcinoma, renal oncocytic hybrid carcinoma, bladder cancer, uterine corpus endometrioid cancer, interdigitating dendritic cell sarcoma, hemangioblastomas, pancreatic neuroendocrine tumors, pheochromocytomas, endolymphatic sac tumors, kidney cysts, and lung cysts.

In one embodiment, a diagnostic method for determining a subject's susceptibility to a disease comprises obtaining nucleic acid sequence data from that subject, detecting the presence or absence of at least one allele of at least one polymorphic marker (or markers in linkage disequilibrium therewith) present within at least one genomic region associated with FNIP1 and/or FNIP2, such as but not limited to genomic regions found in SEQ ID NOs: 1-15, wherein different alleles are associated with different susceptibilities to the disease, and determining a susceptibility to the disease from the nucleic acid sequence data. In one embodiment, the at least one allele associated with susceptibility to a disease is present within an exon of FNIP1 and/or FNIP2 described by SEQ ID NOs: 1-15 that encodes for the FNIP1 and/or FNIP2 protein. In another embodiment, the at least one allele associated with susceptibility to a disease is located within a non-exonic (i.e. non-coding) region of FNIP1 and/or FNIP2 that affects the expression of FNIP1 and/or FNIP2, such as, for example, a promoter, an enhancer, an intron, a 5′ UTR or a 3′ UTR. In another embodiment, a diagnostic method for determining a subject's susceptibility to a disease comprises obtaining a sample, including from tissue, fluids, or other sample containing cellular material, from a subject, analyzing the sample for concentration and/or polymorph(s) of FNIP1 and/or FNIP2 protein, comparing to known and/or calibrated control samples, and determining a susceptibility to the disease. In yet another embodiment, a diagnostic method for determining a subject's susceptibility to a disease comprises obtaining nucleic acid sequence data from the subject, such as for example RNA-seq or cDNA data, detecting the expression levels of at least one transcript that is associated with a sequence described by SEQ ID NOs: 1-15, wherein different expression levels of the transcript(s) are associated with different susceptibilities to the disease, and determining a susceptibility to the disease from the nucleic acid sequence data.

In certain embodiments, the methods of determining risk or susceptibility to a disease, or methods of diagnosis of a disease stated above, can be applied to predict prognosis of a human individual diagnosed with, or experiencing symptoms associated with, the disease. In other embodiments, the methods of determining risk or susceptibility to a disease, or methods of diagnosis of a disease stated above, can be used to assess a human individual for a probability of a response to a therapeutic method and/or modulator used to treat, prevent or ameliorate symptoms associated with the disease. In one embodiment, such methods can be used to select a modulator used in treating a subject with the disease.

In one embodiment, the disease is ALS. In other embodiments, the disease is an inflammatory disease, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, or cancer described herein. In yet other embodiments, the methods of determining risk or susceptibility to ALS, or methods of diagnosis of ALS, or methods of predicting prognosis of a human individual diagnosed with, or experiencing symptoms associated with ALS, or methods of assessing a human individual for a probability of a response to a therapeutic method and/or modulator used to treat, prevent or ameliorate symptoms associated with ALS, can be applied to other diseases, particularly neuromuscular or neurodegenerative diseases, such as, for example, FTLD, Alzheimer's Disease, retinal degeneration diseases such as age-related macular degeneration (AMD), and other TDP-43 proteinopathies disclosed herein; as well as oxidative stress, obesity, anemia or ischemic diseases, such as cardiovascular disease, myocardial ischemia, or peripheral vascular disease.

Kits

Some embodiments also relate to kits and apparatuses for determining susceptibility of a human individual to a disease; or for diagnosing the disease; or predicting prognosis of a human individual diagnosed with, or experiencing symptoms associated with a disease; or assessing a human individual for a probability of a response to a therapeutic method and/or modulator used to treat, prevent or ameliorate symptoms associated with a disease. In one embodiment, the disease is ALS. In some embodiments, the disease is a neuromuscular or neurodegenerative disease, such as, for example, FTLD, Alzheimer's Disease, retinal degeneration disease such as age-related macular degeneration (AMD), and other TDP-43 proteinopathy disclosed herein; as well as oxidative stress, obesity, anemia or ischemic disease, such as cardiovascular disease, myocardial ischemia, or peripheral vascular disease. In other embodiments, the disease is an inflammatory disease, von Hippel-Lindau (VHL) disease, Birt-Hogg-Dube (BHD) syndrome, spontaneous pneumothorax, B cell deficiency, cardiomyopathy, or cancer described herein.

Kits that are useful in any of the methods described herein can comprise of any component that is useful in any of the methods described herein, including but not limited to probes (e.g., hybridization probes, allele-specific oligonucleotides), enzymes (e.g., for RFLP analysis, activity assays), reagents for amplification of nucleic acids, reagents for direct analysis of at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, reagents for indirect analysis of at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, etc. In one embodiment, the kit can include necessary buffers. In another embodiment, the kit can additionally provide reagents for other disease-specific diagnostic methods known in the art to be carried out in conjunction with the methods described herein.

In certain embodiments, the reagents in the kits include at least one contiguous oligonucleotide, such as for example described in SEQ ID NOs: 16-1494, which is capable of hybridizing to a fragment of the genome of the individual containing at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, or markers in linkage disequilibrium therewith. In another embodiment, the reagents in the kits comprise at least two oligonucleotide primers, such as for example described in SEQ ID NOs: 16-1494, which are designed to amplify a fragment of the genome of the individual containing at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, or markers in linkage disequilibrium therewith. Furthermore, the oligonucleotide(s) in the kits can contain mismatches to the fragment of the genome, as is well known to a skilled person in the art. In another embodiment, the kit comprises at least one or more labeled oligonucleotides and reagents for detection of the label. Suitable labels can include but are not limited to a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, or an epitope label.

In some embodiments, the kits and apparatuses include a collection of data comprising correlation data between the at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2 that is selectively assessed by the kit and susceptibility to a disease, or prognosis for the disease, or response to at least one therapy for the disease. In certain embodiments, the kits and apparatuses include a collection of data comprising correlation data between the expression levels of at least one transcript associated with a sequence described by SEQ ID NOs: 1-15 that is selectively assessed by the kit, and susceptibility to a disease, or prognosis for the disease, or response to at least one therapy for the disease. Another set of embodiments relates to methods of use of at least one oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing the susceptibility to a disease in a human individual, wherein the probe is capable of hybridizing to a segment of a nucleic acid containing at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, or markers in linkage disequilibrium therewith. In one embodiment, the segment is 15-500 nucleotides in length. In one embodiment, the kit further comprises a set of instructions for using the reagents comprising the kit. In another embodiment, the kit comprises a set of instructions or guidelines for interpreting the results of a test using the reagents comprising the kit.

A further set of embodiments provides for a kit (also referred to as a pharmaceutical pack and are used interchangeably) comprising a therapeutic modulator and a set of instructions for administration of the therapeutic modulator to a human. The therapeutic modulator can be an antisense modulator, antisense oligonucleotide, other oligonucleotide, a small molecule, an antibody, a peptide, a gene therapy, a nucleic acid vector, or other therapeutic modulator described herein. In one embodiment, an individual identified as a carrier of at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, or markers in linkage disequilibrium therewith, is instructed to take a prescribed dose of the therapeutic modulator. In another embodiment, an individual identified as a homozygous carrier of at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, or markers in linkage disequilibrium therewith, is instructed to take a prescribed dose of the therapeutic modulator. In another embodiment, an individual identified as a non-carrier of at least one allele of at least one polymorphic marker within or associated with FNIP1 and/or FNIP2, or markers in linkage disequilibrium therewith, is instructed to take a prescribed dose of the therapeutic agent.

Computers-Readable Medium and Apparatuses

The materials, methods, and kits described herein can be implemented, in all or in part, as computer executable instructions on computer-readable media. As understood by a person skilled in the art, the various steps of the materials, methods and kits described herein can be implemented as various blocks, operations, routines, tools, modules and techniques, which in turn can be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software. In certain embodiments, hardware implementations can include but are not limited to a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc. In other embodiments, when implemented as software, the software can be stored in any computer readable medium known in the art, including but not limited to a solid-state disk, a magnetic disk, an optical disk, or other storage medium, in a RAM or ROM or flash memory of a computer, processor, hard disk drive, thumb drive, optical disk drive, tape drive, etc. In one embodiment, the software can be delivered to a user or a computing system via any delivery method known in the art, including but not limited to over a communication channel such as the internet, a wireless connection, a satellite connection, a telephone line, a computer readable disk or other transportable computer storage mechanism.

One set of embodiments provides for a suitable computing system environment known in the art to implement the materials, methods and kits described herein, including but not limited to mobile phones, laptops, personal computers, server computers, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, cloud computing environments, and distributed computing environments that include any of the above systems or devices, etc. In some embodiments, the steps of the materials, methods or kits described herein are implemented via computer-executable instructions such as program modules, including but not limited to routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. In one embodiment, the methods and apparatuses are practiced in a distributed computing environment, where tasks are performed by remote processing devices that are linked through a communications network. In one embodiment, the methods and apparatuses are practiced in an integrated computing environment. In both integrated and distributed computing environments, program modules can be located in both local and/or remote computer storage media, including memory storage devices.

Thus, one set of embodiments provides a computer-readable medium having computer executable instructions for determining the effect of administering a modulator to a cell an animal, or a human subject, the computer-readable medium comprising data indicative of the level of at least one protein, nucleotide, marker, or other phenotype, and a routine stored on the computer readable medium and adapted to be executed by a processor to determine the effect of administering the modulator from the data. In certain embodiments, the effect being determined is a change in levels of FNIP1 and/or FNIP2 RNA, or a change in levels of FNIP1 and/or FNIP2 protein, or a change in phenotype such as cell survival, cell morphology, levels of TDP-43 aggregates in the cytoplasm, activation levels of the HIFla-VEGF pathway, levels of angiogenesis, survival of the organism, motor function, respiration, behavior or body weight etc. In certain embodiments, the modulator is an antisense modulator, an antisense oligonucleotide, nucleic acid vector, other oligonucleotide modulator, antibody modulator, peptide modulator, or a small molecule modulator, etc. In one embodiment, the computer-readable medium is used to determine progression of a disease and its response to administration of a modulator described herein to a human subject.

Another set of embodiments provides for a computer-readable medium having computer executable instructions for developing a modulator using at least one computational method described herein, or other computational methods that are known to those skilled in the art, which are also included in the embodiments herein. The computer-readable medium can comprise data associated with a particular nucleotide sequence, SEQ ID NO, or oligonucleotide represented by a specific sample reference number (GiTx ID #), or portion thereof disclosed herein, as well as any resulting polypeptide sequences due to transcription and translation of said nucleotide sequences. The computer-readable medium can also be adapted to be executed by a processor to develop a modulator from said data.

Many modifications and variations can be made in the materials, methods, and kits described herein without departing from the spirit and scope of the invention. Accordingly, it should be understood that the materials, methods, and kits described herein are illustrative only and are not limiting upon the scope of the invention.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are the examples intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications can be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. It should also be appreciated that the examples provide enabling guidance on the use of the combined features of the disclosure to apply such compositions, methods and systems to other uses. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The examples can be implemented in certain embodiments by computers or other processing devices incorporating and/or running software, where the methods and features, software, and processors utilize specialized methods to analyze data.

Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, intensity, temperature, etc.) but some experimental errors and deviations should be accounted for.

Example 1: Antisense Modulation of Human FNIP1 and/or FNIP2 in ReN-VM Cells

In certain embodiments, provided herein are antisense modulators that are antisense oligonucleotides (ASOs), which are designed to target at least one region of at least one transcript produced from the FNIP1 gene (RefSeq Accession No. NC_000005.10 truncated from nucleotides 131639000 to 131799000, incorporated herein as SEQ ID NO: 11) and/or FNIP2 gene (the reverse complement of RefSeq Accession No. NC_000004.12 truncated from nucleotides 158767000 to 158910000, incorporated herein as SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a nucleobase sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary to an equal length portion of SEQ ID NO: 11. In other embodiments, the antisense oligonucleotide has a nucleobase sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary to an equal length portion of SEQ ID NO: 1. In yet other embodiments, the antisense oligonucleotide comprises at least 8, or at least 12, consecutive nucleobases that are at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to an equal length portion of any of the nucleobase sequences of SEQ ID NOs: 16-1494. The following description of particular antisense oligonucleotides and their modifications are provided as examples only and are not meant to limit the scope of the invention.

In some embodiments, provided herein are antisense oligonucleotide modulators that increase the expression or activity of FNIP1 and/or FNIP2. Examples of antisense oligonucleotides that can increase the expression or activity of FNIP1 are described in Table 3. Examples of antisense oligonucleotide modulators that can increase the expression or activity of FNIP2 are described in Table 6. Bolded nucleosides presented in Table 3 and Table 6 indicate nucleosides that are modified with a 2′-MOE sugar modification. For example, GiTx-58, GiTx-59, GiTx-60, GiTx-61, GiTx-62, GiTx-63, GiTx-64, GiTx-65, GiTx-75, GiTx-76, GiTx-77, GiTx-78, GiTx-79 and GiTx-80 are antisense oligonucleotides wherein each nucleoside comprises a 2′-MOE sugar modification. In a preferred embodiment, antisense oligonucleotides that increase the expression or activity of FNIP1 and/or FNIP2 comprise a modified oligonucleotide wherein each internucleoside linkage is a phosphorothioate internucleoside linkage. In a preferred embodiment, antisense oligonucleotides that increase the expression or activity of FNIP1 and/or FNIP2 comprise a modified oligonucleotide wherein each nucleoside contains a modified sugar. In a preferred embodiment, antisense oligonucleotides that increase the expression or activity of FNIP1 and/or FNIP2 comprise a modified oligonucleotide wherein each nucleoside contains a 2′-MOE sugar modification.

In other embodiments, provided herein are antisense oligonucleotides that decrease the expression or activity of FNIP1 and/or FNIP2. Examples of antisense oligonucleotides that can decrease the expression or activity of FNIP1 are described in Table 2. Examples of antisense oligonucleotides that can decrease the expression or activity of FNIP2 are described in Table 5. Bolded nucleosides presented in Table 2 and Table 5 indicate nucleosides that are modified with a 2′-MOE sugar modification. For example, GiTx-51, GiTx-52, GiTx-53, GiTx-54, GiTx-55, GiTx-56, GiTx-57, GiTx-66, GiTx-68, GiTx-69 and GiTx-70 are antisense oligonucleotides wherein each nucleoside comprises a 2′-MOE sugar modification. Underlined nucleosides presented in Table 2 and Table 5 indicate non-modified DNA nucleosides. For example, GiTx-45, GiTx-46, GiTx-47, GiTx-48, GiTx-49, GiTx-50, GiTx-67, GiTx-71, GiTx-72, GiTx-73, and GiTx-74 are gapped sequences comprising a central sequence of unmodified DNA nucleosides flanked by wing sequences on both ends comprising nucleosides with a 2′-MOE sugar modification. In a preferred embodiment, antisense oligonucleotides that decrease the expression or activity of FNIP1 and/or FNIP2 comprise a modified oligonucleotide wherein each internucleoside linkage is a phosphorothioate internucleoside linkage. In a preferred embodiment, antisense oligonucleotides that decrease the expression or activity of FNIP1 and/or FNIP2 comprise a gapped sequence. In another preferred embodiment, antisense oligonucleotides that decrease the expression or activity of FNIP1 and/or FNIP2 comprise a modified oligonucleotide wherein each nucleoside contains a 2′-MOE sugar modification.

The effectiveness of the antisense oligonucleotides in increasing or decreasing the expression of FNIP1 and/or FNIP2 can be tested in mammalian cell lines, such as ReN-VM, HEK-293T, SHSY-5Y, B-35, NRK-49F, 3T3-L1, MEF, etc., according to established protocols. Cells can be seeded at 10,000 cells per well in a 96-well plate, or 50,000 cells per well in a 24-well plate, or 300,000 cells per well in a 6-well plate. After 1 day, antisense oligonucleotides at defined concentrations can be transfected into the cells with or without a transfection reagent such as Lipofectamine. After a 1 day incubation period, the cells can be harvested to obtain RNA. The RNA can be reverse transcribed to form cDNA. The cDNA can be analyzed by quantitative PCR (qPCR) to quantify the relative expression levels of FNIP1 and/or FNIP2 RNA and a control gene, such as beta-actin. The relative expression values of FNIP1 and/or FNIP2 RNA for the different treatment groups can be calculated by normalizing with their respective beta-actin loading control, and presented as a percentage to that of the untreated control (which can comprise cells not transfected with ASOs).

In addition, or alternatively, after a 1, 2 or 3-day incubation period, the cells can be harvested to obtain whole cell lysates. The whole cell lysates can be analyzed by Western blot and probed with an FNIP1 and/or FNIP2-specific antibody, such as described in Table 14. The intensity of Western blot bands can be quantified by FIJI (Image J) and used to quantify the expression levels of FNIP1 and/or FNIP2 protein. The relative expression values of FNIP1 and/or FNIP2 protein for the different treatment groups can be calculated by normalizing with their respective a-Tubulin loading control, and presented as a percentage to that of the untreated control (which can comprise cells not transfected with ASOs).

Example 2: Modulation of Human FNIP1 and/or FNIP2 by RNAi

In certain embodiments, provided herein are antisense modulators that act via the RNAi pathway, such as siRNA and shRNA modulators, which are designed to target at least one region of at least one FNIP1 and/or FNIP2 transcript produced from the FNIP1 gene (RefSeq Accession No. NC_000005.10 truncated from nucleotides 131639000 to 131799000, incorporated herein as SEQ ID NO: 11) and/or FNIP2 gene (the reverse complement of RefSeq Accession No. NC_000004.12 truncated from nucleotides 158767000 to 158910000, incorporated herein as SEQ ID NO: 1). In some embodiments, the strand of the siRNA or shRNA that is antisense to the target has a nucleobase sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary to an equal length portion of SEQ ID NO: 11. In other embodiments, the strand of the siRNA or shRNA that is antisense to the target has a nucleobase sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary to an equal length portion of SEQ ID NO: 1. In yet other embodiments, the strand of the siRNA or shRNA that is antisense to the target comprises at least 8, or at least 12, consecutive nucleobases that are at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to an equal length portion of any of the nucleobase sequences of SEQ ID NOs: 16-1494, wherein thymine bases are replaced by uracil bases. In a preferred embodiment, provided herein are siRNA or shRNA modulators that decrease the expression or activity of FNIP1 and/or FNIP2. In one embodiment, provided herein are siRNA or shRNA modulators that increase the expression or activity of FNIP1 and/or FNIP2.

The siRNA or shRNA modulators can be tested in mammalian cell lines, such as ReN-VM, HEK-293T, SHSY-5Y, B-35, NRK-49F, 3T3-L1, MEF, etc., according to established protocols to evaluate their effectiveness in increasing or decreasing the expression of FNIP1 and/or FNIP2. Cells can be seeded at 10,000 cells per well in a 96-well plate, or 50,000 cells per well in a 24-well plate, or 300,000 cells per well in a 6-well plate. After 1 day, siRNA or shRNA at defined concentrations can be transfected into the cells with or without the use of a transfection reagent such as Lipofectamine. After a 1 day incubation period, the cells can be harvested to obtain RNA. The RNA can be reverse transcribed to form cDNA. The cDNA can be analyzed by quantitative PCR (qPCR) to quantify the relative expression levels of FNIP1 and/or FNIP2 RNA and a control gene, such as beta-actin. The relative expression values of FNIP1 and/or FNIP2 RNA for the different treatment groups can be calculated by normalizing with their respective beta-actin loading control, and presented as a percentage to that of the untreated control (which can comprise cells transfected with a non-targeting siRNA or shRNA).

In addition, or alternatively, after a 1, 2, or 3-day incubation period, the cells can be harvested to obtain whole cell lysates. The whole cell lysates can be analyzed by Western blot and probed with an FNIP1 and/or FNIP2-specific antibody, such as described in Table 14. The intensity of Western blot bands can be quantified by FIJI (Image J) and used to quantify the expression levels of FNIP1 and/or FNIP2 protein. The relative expression values can be calculated by normalizing the FNIP1 and/or FNIP2 bands with its respective α-Tubulin loading control, and presented as a percentage to that of the control, which can comprise cells transfected with a non-targeting siRNA control.

Example 3: Human Stem Cell Models for Evaluating Therapies for Neurodegenerative Disease Such as ALS

In order to evaluate therapies for neurodegenerative diseases such as ALS, in particular to evaluate the modulation of human FNIP1 and/or FNIP2 as a therapy for ALS, several ALS induced pluripotent stem cell (iPSC) lines were obtained from various sources and propagated (Table 7). The cell lines GI-iPSC 2 and GI-iPSC 3, shown in Table 7, are isogenic ALS lines, which have been engineered to contain known disease-causing mutations in ALS genes such as the G298S mutation in TARDBP and the L144F mutation in SOD1 respectively (Hor et al., bioRxiv 713651 (2019)). GI-iPSC 4 through GI-iPSC 9 (commercially sourced), shown in Table 7, are cell lines that are derived from ALS patients with various known and unknown genetic mutations, such as C9orf72 repeat expansion, TARDBP or SOD1 mutations, or sporadic ALS cases with unknown genetic causes. GI-iPSC 1, GI-iPSC 10 and GI-iPSC 11, shown in Table 7, are healthy lines used as controls.

Example 4: Effect of Modulation of Human FNIP1 and/or FNIP2 on Survival of Human ALS iPSC-Derived Motor Neurons

The assay described herein was performed to determine the effect of inhibition of human FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN, on the survival of human ALS iPSC-derived motor neurons. In this specific example, siRNAs targeting and inhibiting FLCN were used to study the effects of inhibition of FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN. Human ALS iPSCs were differentiated into motor neurons over a period of 28 days according to an established protocol (Hor et al., bioRxiv 713651 (2019)). On Day 28, motor neuron cells were transfected with 10 nM of commercially-sourced siRNA that was either validated to inhibit human FLCN (si-FLCN) or that served as the non-targeting control (si-NT) using Lipofectamine. The motor neuron cells were fixed with 4% paraformaldehyde on Days 28 and 31 for determination of motor neuron survival. The fixed motor neuron cells were stained with a specific antibody against the motor neuron marker ISL1, and cellular nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) prior to imaging using the Opera Phenix High-content Screening System (Perkin Elmer). To calculate normalized survival index (NSI shown in Table 8) on Days 28 and 31, the ratio of ISL1+ cells (those cells stained with the ISL1 antibody) to DAPI-stained nuclei was determined and normalized against the ratio of ISL1+ cells to DAPI-stained nuclei for the healthy control motor neurons (BJ-iPS) at Day 28 (Table 8). Results shown in Table 8 are the average of at least 5 technical replicates.

The results indicate that treatment with si-FLCN can increase the survival of human ALS iPSC-derived motor neurons on Day 31, compared to treatment with the non-targeting control siRNA (si-NT) (Table 8). This suggests that inhibition of human FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN (via e.g., an antisense modulator, oligonucleotide modulator, nucleic acid vector, peptide modulator, antibody modulator, or small molecule modulator etc.) can promote the survival of human motor neuron cells. Furthermore, this effect can be observed in iPSC-derived motor neurons representing different ALS sub-types, including but not limited to the isogenic SOD1 (L144F), isogenic TDP-43 (G298S) and patient-derived sporadic ALS line (CS14isALS-Tn16) (Table 8). This finding suggests that decreasing the expression or activity of human FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN via a modulator, such as an antisense modulator, oligonucleotide modulator, nucleic acid vector, peptide modulator, antibody modulator, or small molecule modulator, can be an effective therapy for a broad subset of ALS patients, including, but not limited to, patients with mutations in known ALS genes such as SOD1, C9orf72 and TARDP43, as well as sporadic ALS patients with no known mutations in ALS genes.

Example 5: Effect of Modulation of Human FNIP1 and/or FNIP2 on the Levels of Phosphorylated TDP-43 in the Cytoplasm of Human ALS iPSC-Derived Motor Neurons

The assay described herein was performed to determine the effect of inhibition of human FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN, on the levels of phosphorylated TDP-43 (pTDP-43) in the cytoplasm of human ALS iPSC-derived motor neurons. In this specific example, siRNAs targeting and inhibiting FLCN were used to study the effects of inhibition of FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN. Human ALS iPSCs were differentiated into motor neurons over a period of 28 days according to an established protocol (Hor et al., bioRxiv 713651 (2019)). On Day 28, motor neuron cells were transfected with 10 nM of commercially sourced siRNA that was either validated to inhibit human FLCN (si-FLCN) or that served as the non-targeting control (si-NT) using Lipofectamine. The motor neuron cells were fixed with 4% paraformaldehyde on Days 28 and 31 to determine the levels of pTDP-43 in the cytoplasm. The fixed motor neuron cells were stained with a specific antibody against the motor neuron marker ISL1, counterstained with DAPI, and also co-stained with a specific antibody against TDP-43 phosphorylated at Ser 409 or Ser 410, prior to imaging using the Opera Phenix High-content Screening System (Perkin Elmer). The number of pTDP-43 foci (spots) per unit area of cytoplasm, or the intensity of pTDP-43 staining in the cytoplasm, were quantified and used as a proxy for the levels of cytoplasmic pTDP-43. The levels of pTDP-43 in the cytoplasm on Days 28 and 31 for the different treatment groups were normalized against the cytoplasmic pTDP-43 levels in healthy control motor neurons (BJ-iPS) at Day 28 (Table 9). Results shown in Table 9 are the average of at least 5 technical replicates.

Over 97% of all ALS cases (both sporadic and familial) display pTDP-43 positive aggregates in the cytoplasm of affected neurons; cytoplasmic pTDP-43 aggregates have been associated with ALS pathology (Prasad et al., Frontiers in Molecular Neuroscience, 12:25 (2019)). The results shown in Table 9 indicate that cytoplasmic pTDP-43 levels are elevated to over eight-fold at Day 28 in all ALS iPSC-derived motor neurons compared to the healthy BJ-iPS control, including, but not limited to, the isogenic SOD1 (L144F), isogenic TDP-43 (G298S) and patient-derived sporadic ALS line (CS14isALS-Tn16). This finding suggests that the ALS iPSC-derived motor neurons can replicate important pathological features of ALS and can serve as a relevant disease model. Treatment of the ALS iPSC-derived motor neurons with si-FLCN but not si-NT reduced the levels of cytoplasmic pTDP-43 aggregates in those cells (Table 9). This suggests that decreasing the expression or activity of human FNIP1 and/or FNIP2 leading to a decrease in expression and/or activity of FLCN via a modulator, such as an antisense modulator, oligonucleotide modulator, nucleic acid vector, peptide modulator, antibody modulator, or small molecule modulator, can reduce the levels of pathological cytoplasmic pTDP-43 aggregates and potentially improve disease outcomes for ALS patients.

Example 6: Identification of miRNA Modulators

miRNA modulators that are capable of targeting nucleic acids encoding FNIP1 and/or FNIP2 can be identified using various databases such as for example, miRDB, which is a database that contains predicted miRNA-gene target interactions (Chen et al., Nucleic Acids Research, 48: D127-D131 (2020)). In some embodiments, provided herein are miRNA modulators that can inhibit or decrease the expression or activity of FNIP1 and/or FNIP2. In other embodiments, provided herein are miRNA modulators that can increase the expression or activity of FNIP1 and/or FNIP2. miRNA modulators, their nucleobase sequences, and examples of predicted target sites in SEQ ID NO: 11, are disclosed herein in Table 10. miRNA modulators, their nucleobase sequences, and examples of predicted target sites in SEQ ID NO: 1, are disclosed herein in Table 11. In certain embodiments, miRNA modulators, or portions thereof, are at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more particular miRNA sequences disclosed in Table 10 and/or Table 11. In one embodiment, a miRNA disclosed herein can be encoded by a nucleic acid vector and delivered into a cell, an animal or human, in order to inhibit the expression or activity of FNIP1 and/or FNIP2.

Example 7: Identification of Small Molecule Modulators

Small molecule modulators that are capable of targeting nucleic acids encoding FNIP1 and/or FNIP2 were identified using methods of development described herein. In one embodiment, the target regions, or structurally defined regions of nucleic acids encoding FNIP1 and/or FNIP2, were first identified using computational tools well known in the art that predict the existence of a target region, and/or that predict the stability or structure of a nucleic acid region. Next, a high-throughput computational screen was performed to identify small molecule exemplars and small molecule scaffolds (otherwise known as pharmacophores) that can bind to each target region of a nucleic acid encoding FNIP1 and/or FNIP2. Small molecule exemplars with excellent predicted dissociation constants against a target region of a nucleic acid encoding FNIP1 (the lower the better) were identified and listed in Table 12. Small molecule exemplars with excellent predicted dissociation constants against a target region of a nucleic acid encoding FNIP2 (the lower the better) were identified and listed in Table 13.

Small molecule modulators that are capable of targeting FNIP1 and/or FNIP2 proteins were identified using methods of development described herein. Briefly, a high-throughput computational screen was performed to identify small molecule exemplars and small molecule scaffolds (otherwise known as pharmacophores) that can bind to the FNIP1 and/or FNIP2 proteins. X-ray crystal structures of the FNIP1 and FNIP2 proteins were obtained from the Protein Data Bank (PDB). The cavities on the surfaces of FNIP1 and FNIP2 that are amenable to small molecule binding were identified using established tools such as Autodock. Molecular docking was performed using computational databases of small molecule ligands, which include small molecule exemplars and scaffolds, to the identified cavities on the surfaces of the FNIP1 and FNIP2 proteins. Molecular docking was also performed using computational databases of small molecule ligands to the entire surfaces of the FNIP1 and FNIP2 proteins. Small molecule exemplars with excellent predicted dissociation constants with the FNIP1 protein (the lower the better) were identified and listed in Table 12. Small molecule exemplars with excellent predicted dissociation constants with the FNIP2 protein (the lower the better) were identified and listed in Table 13.

In certain embodiments, small molecule exemplars described in Table 12 and/or Table 13 can be used as modulators to increase the expression or activity of FNIP1 and/or FNIP2. In some embodiments, small molecule modulators that are similar in structure to the exemplars described in Table 12 and/or Table 13 can be used as modulators to increase the expression or activity of FNIP1 and/or FNIP2. In other embodiments, small molecule modulators that have a Tanimoto index of at least 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00 compared to at least one exemplar or scaffold described in Table 12 and/or Table 13 can be used to increase the expression or activity of FNIP1 and/or FNIP2.

In other embodiments, small molecule exemplars described in Table 12 and/or Table 13 can be used as modulators to inhibit or decrease the expression or activity of FNIP1 and/or FNIP2. In some embodiments, small molecule modulators that are similar in structure to the exemplars described in Table 12 and/or Table 13 can be used as modulators to inhibit or decrease the expression or activity of FNIP1 and/or FNIP2. In other embodiments, small molecule modulators that have a Tanimoto index of at least 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00 compared to at least one exemplar or scaffold described in Table 12 and/or Table 13 can be used to inhibit or decrease the expression or activity of FNIP1 and/or FNIP2.

Example 8: Identification of Antibody or Peptide Modulators

Antibody modulators that are capable of targeting FNIP1 and/or FNIP2 can be identified from commercial sources and are disclosed in Table 14. In some embodiments, such antibodies can be capable of increasing the expression or activity of FNIP1 and/or FNIP2. In other embodiments, such antibodies can be capable of inhibiting or decreasing the expression or activity of FNIP1 and/or FNIP2. Furthermore, antibodies, antibody fragments, monobodies, or other peptide modulators comprising a CDR that is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% similar to the CDR of at least one antibody described in Table 14, as assessed by sequence alignment or other scoring methods known in the art, can be capable of increasing or decreasing the expression or activity of FNIP1 and/or FNIP2, and are included herein.

Tables

TABLE 1 Example antisense oligonucleotide modulators targeting SEQ ID NO: 11 Target start Target stop site with site with reference to reference to SEQ ID SEQ ID Binding SEQ ID No ASO sequence NO: 11 NO: 11 Score NO 1 CAGCTATGTACATATCCAAC 157170 157189 -26 16 2 TAAGTGATCATCCCCAGAGT 157072 157091 -33.1 17 3 GACCACACTTTAAAGTAGCA 156769 156788 -26.3 18 4 GGAAGCAGTGGGGAGGAACA 156654 156673 -30.5 19 5 TTGATGGTTAAGGAGACACC 156468 156487 -27.4 20 6 GGCCCGCAAATGTATGAACT 156083 156102 -31.3 21 7 ACTGGGACTAGGTAAAAGGT 155902 155921 -25 22 8 GTCTCTTAGACCAGATAAGC 155568 155587 -25.3 23 9 CTTACAAGAGTTTGGTTCCA 155460 155479 -26.7 24 10 TACACTTTTGGAAGGGAGGG 155363 155382 -27.7 25 11 CTATGGGTTCATGAGGGTTG 155258 155277 -29.1 26 12 AAGGTTGGCTTTACTTATAT 155030 155049 -25.7 27 13 GGGTGGATAACATAACACAT 154856 154875 -28 28 14 CGTACACTTTGGCTTTTTCA 154563 154582 -24.9 29 15 GCATGTTGTGTCTGCTTCCT 154364 154383 -32.1 30 16 GTGCAACATATGGAGAGTGA 154283 154302 -32 31 17 TCTGTACTGAGGACATGAAC 154182 154201 -27.8 32 18 GGGAGGTTGAGGCTGAAGTC 153735 153754 -33.5 33 19 GTACCAACAGCAGGGCAACA 153709 153728 -32.3 34 20 AGATAGTAGCCTAGCAAAGA 153457 153476 -31.6 35 21 GGTTTATAGTAGCAAAAAAC 153249 153268 -27 36 22 GGCAAGTTGTACAAGGACAC 153129 153148 -31.8 37 23 CTGGACCAACAGTGTGGTAC 152796 152815 -29.9 38 24 GTGGTTTGACATACAGTAGG 152589 152608 -28.3 39 25 GAAATAGGGCTATATTATGC 152413 152432 -30.3 40 26 GTGGAAGGCAGGGCAGATAC 152224 152243 -34.7 41 27 GATAACAGCCTCCAGCTTCT 152016 152035 -31.4 42 28 CTCAGGTATTCAGACAGCAT 151847 151866 -33.4 43 29 GAGGAACCAAGAACCAGGTC 151765 151784 -26.8 44 30 CATGCGCACCACACCCGGCT 151364 151383 -31.5 45 31 AACATTCTGGGAGGTGGAGG 150902 150921 -35 46 32 GATGCTAAACATGTTTGAGG 150582 150601 -23.7 47 33 GCTAAACATTAAATTCATAC 150542 150561 -24.7 48 34 TTCTCTTCTTACCCGCAGCC 150171 150190 -35 49 35 GCCTATTTGTGGTACCTCAT 150112 150131 -26.9 50 36 GGGTTCCAAGGTCACCACAT 149698 149717 -29 51 37 TCCTGTTGAGTTGTAGGAGT 149469 149488 -27 52 38 AGTTCATTTCTGTACGTGGC 149179 149198 -33.1 53 39 TTGGTCTATATACCTGTCCT 149012 149031 -28.5 54 40 TTTGAGTAGTAATGATATCC 148780 148799 -29.7 55 41 GGTGCTATTGTAAGTGGAAT 148611 148630 -28.5 56 42 TCTGTGGGATCTTTATGTTT 148469 148488 -31.2 57 43 GGGAAAGTGGGTCTTATTGT 148315 148334 -32.2 58 44 GCTGGGAGATGTTGCATGAC 148031 148050 -33.8 59 45 GGCTGGGGCAGGAGGAACAC 147830 147849 -35 60 46 CCTCCCAAAGATCCTATCTC 147352 147371 -32.8 61 47 GCTACTGGCCACTTGAACAG 147066 147085 -34 62 48 CAGACAGCTTCTGCTATTGG 147020 147039 -30 63 49 GTCTACTATGTGCCAGGCAA 146686 146705 -28.6 64 50 AGGTTCAAGCCATTCCCCTG 146534 146553 -28.2 65 51 TATAATGCAGGCACTTTGGG 145703 145722 -33.7 66 52 ATAATGCAGGCACTTTGGGA 145702 145721 -34.4 67 53 TACTACTGGCCAAATGGAAT 144690 144709 -32.6 68 54 TTAATGTTACTACAATTAGG 144600 144619 -24.4 69 55 CGCAGTGAAAGGGACATAGG 144307 144326 -35 70 56 AAGAAGAGAATGGCAAAGGA 144150 144169 -28.5 71 57 TACAGGTCCACTTAGATGCC 143916 143935 -31.4 72 58 GGATGCATGCCACAGTAGTG 143760 143779 -32 73 59 CTACTGGGGAGGCAGAGGCG 143723 143742 -28.6 74 60 GCAGTTCCGCTCTCCAATAA 143331 143350 -30.2 75 61 GGTGTTTAAAGGTTCTCATG 142876 142895 -30.1 76 62 GGACCATAGATTAAGTAGCT 142709 142728 -29.5 77 63 ATGTGGGTACAGGGTTGCTG 142527 142546 -35 78 64 AAGGAACAGGAGCAAGCACC 142191 142210 -32.9 79 65 CCATGCCTGGCTCATTTGTG 141825 141844 -32.1 80 66 GTACCTTGCCAGTAAGTGAC 141650 141669 -34.5 81 67 TGATTGTCAGTGCTATGGAA 141466 141485 -32 82 68 ACAGACACCATACCAGAGGA 140892 140911 -32.6 83 69 CCTGGGGTATGGGGCCAAGC 140600 140619 -33.3 84 70 GGGGTGGCTTTCAGGGGGGC 140261 140280 -35 85 71 GCGATGGACGATGGAGAGGC 139924 139943 -29.3 86 72 CCATGCTCAGGAAGGAAGGC 139672 139691 -35 87 73 GCTCAGGAAGGAAGGCTGGA 139668 139687 -32.8 88 74 GTAGCTGTGCTCGGTGAGGA 139458 139477 -33.5 89 75 GGATTCAGGGGGACCTCAGT 139200 139219 -33.2 90 76 AGACGGCCCGGGGGCATCGT 138956 138975 -29.3 91 77 GGCAGGTGTGGATGGGCGGC 138855 138874 -35 92 78 CCAACAAATCAGCCGTAAGC 138455 138474 -30.7 93 79 CTCTAAACTGGTCTGGTGGG 138027 138046 -35 94 80 TGCTCAGGGATCTTGCTTTG 137811 137830 -34.6 95 81 GTACATCTAGAGGGGATCTC 137726 137745 -30.1 96 82 GCTGAGCAAAGTGACTAGTG 137467 137486 -34.1 97 83 TAATTTGATGGTCTCTCGCC 137403 137422 -30.6 98 84 TGAGGCTGTAGTGATTCTGG 137089 137108 -35 99 85 TCCGAAAGGGCTGGGAGTCT 136885 136904 -35 100 86 GGTGGGTGGCTTGGGTTCAA 136412 136431 -30.8 101 87 TCCCTTCCATGCACAGCTTC 136323 136342 -31 102 88 GGCTGGCCTGGGGATATTCT 135947 135966 -32.1 103 89 GAAAGCTCCAGCCTTCCCTT 135676 135695 -27.8 104 90 GCTGGATGCTTTAAGGTGAT 135373 135392 -28.4 105 91 GCTGGGAAAAGTCATGGGTT 135251 135270 -31.5 106 92 GAATGTGGTTTTGATGGGAG 135092 135111 -31.6 107 93 GATGGGAGGCTATAAGAAGG 135080 135099 -35 108 94 GTAGAGGGTTTGTGAAGGGT 134947 134966 -35 109 95 GAACAAGATTTTTGGGAGGC 134602 134621 -32.2 110 96 TGGGCAAGCTGAGATGGGAG 134455 134474 -33.8 111 97 CACACTATGTTAGTATGGGG 134248 134267 -28.5 112 98 TAATCATAACAAGAAGATGC 133903 133922 -24 113 99 CCTAGAGTAAGTATTCTGTC 133791 133810 -25.7 114 100 ATGCCTGGAGGAAGTTTCTG 133709 133728 -31.8 115 101 GGCTTTCTTCACATTTCAAC 133648 133667 -27.7 116 102 GCCTGGAGTGCAATGGTGAA 133348 133367 -33.5 117 103 GAGAAGGAAAGACTAGAAAC 132633 132652 -29.9 118 104 CTCTGAGCCATATCTGAAAC 132366 132385 -27 119 105 TCAGCAGGAGACAGCAATGT 132176 132195 -30.7 120 106 GGCCTTATTTTCTCTGTTGC 131788 131807 -29 121 107 CCCTTGGAGTACTGGCACTA 131720 131739 -29.5 122 108 TTAGGTAACTTTCTACAGAC 131528 131547 -26.5 123 109 TGGTTGTCTTGCTCTGTTGC 131372 131391 -33.5 124 110 GAAAATCAGCACGGACATAA 130927 130946 -29.6 125 111 GAGGTGAACAAAGTAGGTTC 130783 130802 -30.2 126 112 GGTTTGGGGGCTGAAAAGTC 130250 130269 -28.2 127 113 GCAGGACAATTTGGCCCACT 130129 130148 -26.6 128 114 GGATGCAGATGGTTTCACTA 129745 129764 -31 129 115 CCTGATATCAGAGCTAGACA 129639 129658 -26.3 130 116 ACTCATTCATGATTAAGGGA 129350 129369 -25.5 131 117 GGCAACGATGTCCATGGTCA 129133 129152 -27.3 132 118 GGGGCAGCGGGGACAAAAGG 129044 129063 -35 133 119 CCCCACTTCTATATGTAAGC 128871 128890 -29.5 134 120 GATACAAAGATACGGGCACT 128673 128692 -30.4 135 121 GGAACCTCTCATCACTCCCA 128481 128500 -30.4 136 122 GGCAATGTTGGGCTGAACAG 128393 128412 -32.2 137 123 TCAAGAGTAAAATCATCGTT 128163 128182 -25 138 124 TTTCCTCTTAGTCTTGGCCA 127855 127874 -31.1 139 125 TGAAAACAAGAGAGCTATCC 127598 127617 -27.2 140 126 ACCTGTATCTTCACTTTCAC 127394 127413 -27.3 141 127 TCTTGGAATGTCCCATTCAG 127343 127362 -27.9 142 128 GCTCATGCTTTCTGGGTCTG 127037 127056 -31.1 143 129 GATCAATAGCCCCTCTTTCT 126915 126934 -31.2 144 130 CACTCTGCCATGTTTCCTCT 126663 126682 -29.4 145 131 GCCTGACTCTGACAATGCAC 126632 126651 -27.8 146 132 GTGACAAGGACATACTCTGA 126298 126317 -32.4 147 133 ACGGGAGAGCCAATAGCGCC 126088 126107 -35 148 134 CACTGTAATCCTGGCTGGAG 125902 125921 -28.1 149 135 GGGTTTTCTGGTTTCCTCTT 125539 125558 -29.9 150 136 GGAGAGGAAAGGAGTAGTGG 125317 125336 -35 151 137 CCTCTAGCCAGCACCCCACC 124868 124887 -35 152 138 TGGGGAGGTGGAAGCAGGCT 124511 124530 -35 153 139 TCTCTTCTGGCTAACACACT 124077 124096 -31 154 140 GATGTCCTGTCCCTTTCCTC 123802 123821 -35 155 141 GAAAGTAGGCCAGTGGCTGC 123308 123327 -28.4 156 142 GGGATGGTGGAAGAGGAACA 123285 123304 -35 157 143 GTTACGCGCCTGTAGTCCCA 122357 122376 -30.2 158 144 GCACCCTGGTACAATCCCTC 121800 121819 -35 159 145 GGCAGACATGAAAGGACAAG 121426 121445 -29.5 160 146 AGAATGCAGGAGGTAAAAGC 121389 121408 -28 161 147 GGAGAAGAGAGAGGAAAGGA 120988 121007 -35 162 148 AGAGAGGAAAGGATGAGTAT 120981 121000 -33.1 163 149 CAGCAGGTCCTTACATTTCC 120774 120793 -26.6 164 150 CCATCACGCCTGGCCATCCT 120287 120306 -32.4 165 151 TCACAAAAAAATGGGCAGCC 120216 120235 -31.9 166 152 TTTCTGGAGTCCCCGACATC 119886 119905 -32.1 167 153 GCCAGCTTGGTCCTTACATC 119729 119748 -30.8 168 154 GCTCTCCCCACCAGTGACTG 119602 119621 -33 169 155 AGCCCATTCCAGCTCCACAT 119498 119517 -28.5 170 156 CTTAAGGACAGCGAGAACAG 119320 119339 -30.7 171 157 GCTAGTCCTGTGACTTGTAC 119170 119189 -28.6 172 158 GGAAACCTGCACTGTCTGAA 118937 118956 -34.8 173 159 GTGGCTGACGGCTGAGATTT 118820 118839 -31.9 174 160 GTAACACCTAGGAGCCAAAA 118725 118744 -28.5 175 161 CAGGTGTCCAGATACGATGG 118476 118495 -27.5 176 162 GAAGAACCAGCTGGGTGTGG 118351 118370 -30.2 177 163 GGTCATAGTATCACAGAGGG 118083 118102 -35 178 164 GCAGTTGAATGGCCCCGAGG 117860 117879 -34.3 179 165 CATGGGATTGTGGGGGGCGG 117754 117773 -35 180 166 GAGAAAGCCTCCAAGCAGAG 117538 117557 -29 181 167 GACTCCAGAGGAAAACAGAA 117473 117492 -32.4 182 168 GGATTCCAGAGAGAAAACAG 117202 117221 -31.8 183 169 TACCAGAGCCGAAGAACATG 117154 117173 -32.5 184 170 GTGTCTGGGAAGAATGGGGT 116961 116980 -35 185 171 GTCATTGCACAGAGCCAGAT 116759 116778 -31.1 186 172 GGCCTCAGAGTGTTATCAAG 116653 116672 -31 187 173 CATGGCAAAATCCCGTCTAC 116448 116467 -32.8 188 174 GCCCGGACAATGAAAGACTC 116109 116128 -29.6 189 175 TCCTTCCTCTTCTCAACTGC 115929 115948 -31.3 190 176 TGATCCGAGGTTGGCTGATT 115276 115295 -35 191 177 TGTGCCTCTCTGTTTAAGGA 115065 115084 -33 192 178 GGAAGCCATTTTTAACAGCA 114886 114905 -28.5 193 179 TAGGAACATATGCGTCAGGC 114739 114758 -31.2 194 180 GGGGTTACAAATAAATGTTA 114653 114672 -29.2 195 181 GCATGCTGTAAATTCCAGGC 114412 114431 -28.5 196 182 TGGTGACTTCAGCTGGATCT 114230 114249 -34.2 197 183 TTCCTCAGAGCCCAAGCAGA 114197 114216 -28.9 198 184 GTCTGGATTTCGATAAAACG 113942 113961 -27.7 199 185 CCAGTGAGCTATCCTCTGCC 113720 113739 -28.8 200 186 GAGCCACTGCACCTCGCTCC 113246 113265 -31.7 201 187 GCTGACTCTGACATCCCTGC 112755 112774 -34 202 188 CCTGCCAAGCTTCAGCCTCC 112531 112550 -31.4 203 189 ATGGGGTACAATGTGTAATA 112388 112407 -29.2 204 190 GCAATCCTCTCACCGTGGCC 112181 112200 -33.5 205 191 GACAGCAGTATCTGAATGGT 111998 112017 -29.8 206 192 AATGATCCACCTACCTTGGC 111641 111660 -31 207 193 CCAAACTGCGGACAGAGGGA 111375 111394 -31.8 208 194 GACAAAAACTCCTAGGGACC 111198 111217 -32.7 209 195 CCTTCCTCGCTGGCCTCTGG 110974 110993 -34.2 210 196 CCATATCAACATAGCTCCAG 110810 110829 -27.2 211 197 GGGAAAGTGAGGCTTCTGGC 110669 110688 -34.4 212 198 TAATGCAACAGCGAGATGGA 110336 110355 -28.9 213 199 GGAGCAGGAATGAAGAATGC 110263 110282 -32.4 214 200 ACCATAGACGCAGGAGGCTC 110013 110032 -34.8 215 201 CAACCTTACCTATAGAAGAG 109885 109904 -27.9 216 202 GAGACTGGAGAAAAATGGTT 109818 109837 -28.1 217 203 TAAGGGATGTGATGTGAGGG 109461 109480 -35 218 204 GAAGTATGTGCACTCCCTGT 109419 109438 -29.7 219 205 GAGGAGAGAAAAATGGAAGC 109297 109316 -35 220 206 GGTATAGAAGCCGGGTGCAG 109051 109070 -33.4 221 207 ATAGAAGCCGGGTGCAGTGG 109048 109067 -35 222 208 AGCAGAGGTCAGAACAGTGA 108250 108269 -30.2 223 209 AGGCGGAAAATTAGTAAGGA 108030 108049 -27.8 224 210 CCATGCTGACAGGGAACATT 107919 107938 -25.9 225 211 GGTCAAAGAAGACATCTTGA 107729 107748 -24.5 226 212 TAGTAAAATCTAGAGCAGAA 107507 107526 -27.3 227 213 AGAAACAAAAGAGGGGTATC 107355 107374 -30.3 228 214 TTATCTACTCCTGGCAAACA 107159 107178 -29.5 229 215 GGGAGCAAGGTCAAGATGTG 107036 107055 -31.3 230 216 CTCCCCCTGACCCCCCCAAA 106675 106694 -29.5 231 217 GAGAATCTCAGCAAGTTATT 106470 106489 -25.9 232 218 GATAGCATGGGGATTGGGAG 106305 106324 -35 233 219 TACTGGAATATTGCTAAGAG 105889 105908 -29.3 234 220 GAACAGAACAGGGAGCCCCC 105588 105607 -34.8 235 221 GGTGCTGCAACGACTGGACA 105479 105498 -29.8 236 222 GGCTAAAATTGACAAACTGA 104937 104956 -25.5 237 223 TAATTGCTGGTGGGAATGCA 104872 104891 -26.9 238 224 TCAAGCATACAACATTCTGG 104468 104487 -24.4 239 225 ACAGGGGATTTTTAGGGCGG 104364 104383 -33.7 240 226 GGGAAAACTGTAGGTTGGGG 103817 103836 -35 241 227 GTCCCCCACTCCCACTCTCA 103552 103571 -32.2 242 228 TCACCAACTCCTAAAACCGA 103379 103398 -27.4 243 229 GAGAGAGATACAGCCTATGC 103287 103306 -35 244 230 GGCAGGCCTAAACCATTAAC 103017 103036 -29 245 231 GAAGCCAAAGTAGACTGTAC 102778 102797 -23.2 246 232 AAGTTGCTCTAAATTCCCTC 102742 102761 -31.9 247 233 GAGTGAAGATGCCAGTAAAC 102559 102578 -29.2 248 234 GTTAGCATGCTCCATATAAT 102347 102366 -25.3 249 235 TATATATATGTATGGCATGC 101944 101963 -28.2 250 236 ACTCCAAGATGTGATGGCCC 101872 101891 -29.3 251 237 GCCTGGCCCAGATACATATT 101565 101584 -28 252 238 GGGGAAGCACTGCTGTAGGT 101331 101350 -35 253 239 TGCACTTGCCCAAGATCACC 100832 100851 -31.2 254 240 CTAAAGGAGGAAAGATTGAG 100714 100733 -30.9 255 241 GCAGTTGTTGGGGCTTCAGG 100573 100592 -30.6 256 242 GCTAGAACAAGGGTCTTTCC 100327 100346 -28.4 257 243 CTGGCATCAGCTGATCTCCG 100014 100033 -30 258 244 TGGCATCAGCTGATCTCCGG 100013 100032 -29.1 259 245 TCCCAAACAGAACAAGACAT 99676 99695 -24.5 260 246 TCAGTAACAACAACTCATGG 99647 99666 -26.8 261 247 GGGATCAGGATTAGGATATG 98635 98654 -31.3 262 248 ACACCCAAAAGCTTCAGAGG 98471 98490 -26 263 249 GAAAACTGGCCCTAGAAGAT 98225 98244 -25.5 264 250 ACATTTTAGGTCGGGGTCCC 98003 98022 -32.5 265 251 TGGGTCGAGCAAGCAAGTGA 97936 97955 -35 266 252 TTAATGAGTTCTATAATGCC 97628 97647 -26.9 267 253 CATCCTTAGTCACTAATGAG 97334 97353 -23.8 268 254 CCTCTCGTCTCTTACTCCAG 97264 97283 -34.5 269 255 GTACCTCTCACTATTCCTCT 97074 97093 -29.9 270 256 GAGCTCAAGCACCCCTCCCA 96712 96731 -34.4 271 257 GCCATGTTGCTCAATCTGAG 96605 96624 -26.6 272 258 TCCAAATGAAACTTCTTCCC 96366 96385 -31 273 259 CCTTCGCCTCTGCTGGTTCC 96118 96137 -34.4 274 260 TTGCCTGTCCTCTCTTGCCA 96044 96063 -35 275 261 GGTTTGCGTATTCATCATCT 95724 95743 -31.9 276 262 TGTCCTTCTTCCTCCACTGA 95607 95626 -31.1 277 263 GCACCTTGGCCTCGTAATGC 95478 95497 -33.5 278 264 CCTGACTAGCCTATAAAAAT 95328 95347 -31.7 279 265 TCAATCTATTTGCATCCACC 95060 95079 -31.8 280 266 GCTTACCTGTTCTATTGCAC 94923 94942 -28.9 281 267 GGCTATTCTTTCAGGTTCAT 94557 94576 -26.4 282 268 TATAGATGAGTCTGGTACAA 94414 94433 -26.4 283 269 GGAGTAAAAAGGCATCCCAC 93997 94016 -31.4 284 270 GGGCGACTCAAAAGGGAAAG 93533 93552 -32.6 285 271 GAAAATGGGGACTCGAACAG 93219 93238 -28.9 286 272 AGAATGGTGGTTACAAGAGG 92919 92938 -35 287 273 GAGTTTCAGCTGGGAAGAGG 92859 92878 -35 288 274 GTGAGGAGTTTGGGGAAGGA 92486 92505 -34.5 289 275 CTGAGAGATGCTGTTAAGTC 92445 92464 -27.3 290 276 TATCCCTTTGGCCACTGGAT 92160 92179 -31.7 291 277 GAGGTCATTTACAAGTATTC 92009 92028 -30.6 292 278 GCTTGAGGTGTTAAGAGCTA 91772 91791 -33.3 293 279 AGGAGATTTTCATCAGGCTT 91728 91747 -30.8 294 280 CAAGATTGGGTATGTACAAC 91333 91352 -27.3 295 281 TGGGTATGACTTCAGTGAAA 91307 91326 -30.6 296 282 GCCAGGGAAGACTAAATATC 91056 91075 -28.6 297 283 GGTCGGCAACCCTAGCTATC 90907 90926 -29.4 298 284 GCTGCTCTCTGGGCCCCAAC 90561 90580 -33.4 299 285 GTGTGGTGCTCTTTCGCTGA 90407 90426 -31.1 300 286 GGTGCTCTTTCGCTGAAGCT 90403 90422 -31.1 301 287 TACTACAGTTTCTAAGCAGT 90074 90093 -27.9 302 288 GCCACTGTCTCTGTCCTCAT 89768 89787 -30.7 303 289 GCTCTCTTCCAGGCATGTCC 89745 89764 -32.6 304 290 GAAGGGCTAAATTGATACCA 89108 89127 -30.6 305 291 TCTGAGAACAATGAAGAGTA 89087 89106 -27.5 306 292 GGTACAAAAGCTACTCAGCC 88770 88789 -31 307 293 AGACCTACATGGCAAAAACG 88324 88343 -30.3 308 294 GCCACAATGCGTCAGGGAAA 88272 88291 -33.6 309 295 TTGAAAAAGTACGATAATGT 87998 88017 -25.6 310 296 GCAGCGAGGAGAAAACCACC 87900 87919 -28.4 311 297 CACTGTGGAAGGAGGAACAC 87742 87761 -27.9 312 298 GTTCAAGAGAAGTGGCTGCC 87689 87708 -34.5 313 299 CCCTCCCCATGAGCCAGGAC 87369 87388 -35 314 300 AGCTAGGACCCCAATCCCCC 86767 86786 -35 315 301 CTTCTTCAAAAACCTCCTTC 86744 86763 -30.3 316 302 CATGAAAGGGGGTATTAGTG 86347 86366 -29.6 317 303 GCCACCAAAAGCCTTCCTGT 86084 86103 -30.4 318 304 TTTCCCCCTCAGTCTTAGTC 85961 85980 -35 319 305 CGCGCCCGGCCTCGTACCCC 85647 85666 -35 320 306 ACAACTAACTGGTCAACTTC 85490 85509 -27.8 321 307 CTGATTTCTTCTATTTCCCT 85318 85337 -28.3 322 308 TCCTTTTGCCTCACCTCTTC 85238 85257 -32 323 309 GGGCCTTTTCTCAATCCCAC 85089 85108 -28.2 324 310 AGGACACATAGCTGATAGGC 84890 84909 -33.3 325 311 CTCTCAGCCTTTCCCAGCAT 84656 84675 -30.8 326 312 TGCACTGGTGCCCAACTACA 84524 84543 -33.3 327 313 TTACCCCTACTCTGTGCTCC 84413 84432 -35 328 314 TTCTTCCCCTAACCATCTGA 84304 84323 -35 329 315 CACTATAAATCCATTGAGGC 84124 84143 -28.1 330 316 ATGAATTCCTTTTGGCTGGG 83792 83811 -32.2 331 317 TCCCTTGGCTACTACATAGC 83058 83077 -30.3 332 318 AGTATCGCAATACAGCAGCA 83023 83042 -31 333 319 TTTATGGTTATGGAAGACAG 82801 82820 -29 334 320 GCCTCTTCCACTTACAGTAT 82548 82567 -30.2 335 321 GACTATCTTGTAGCCTATGG 82339 82358 -25.3 336 322 CTTGTAGCCTATGGAGGGTG 82333 82352 -31.6 337 323 GCCTGGAGTGCCAGATCTAG 82165 82184 -28.4 338 324 GGGAGAGGTGGAAGCTAGGA 81444 81463 -33.9 339 325 GGTGGAAGCTAGGAGGAAGA 81438 81457 -34 340 326 GTCAGCCGGGCGTGGTGGCC 80940 80959 -33.8 341 327 GTCTGGAGTAGCTTCCTTGC 80657 80676 -30.4 342 328 CTCTGGGGTCTTCCACTGCC 80558 80577 -34.6 343 329 ATGTGCAATATCAGTATGGC 80396 80415 -29.4 344 330 GCCCTAAGACGGAAATCCAT 80246 80265 -29 345 331 GCTGTGGAGGGGAACTATGA 79935 79954 -33.2 346 332 GAAAGAGAGAGACCAAAACT 79906 79925 -30.1 347 333 CATGTTAAATGGAGTATGAC 79782 79801 -26.2 348 334 TGGCATCAGAAGAGCACCGA 79589 79608 -28.8 349 335 GTCAGACCAGTTCCCTTTAC 79308 79327 -27.7 350 336 TACTCACGGGCATTTAAGTC 79291 79310 -28 351 337 CCATAAATGGGGCAATTTAT 79119 79138 -27.3 352 338 GGCTTACATGTATGACCTGA 78623 78642 -27.9 353 339 CCAAAGCACAGGTAACAACA 78525 78544 -28.5 354 340 TATGGAATGGGAAAAGATAA 78422 78441 -28.8 355 341 GACAACAAGTGGTGAGGATG 78154 78173 -34.6 356 342 TGGTAGTTACCAGGGGCTGG 77667 77686 -35 357 343 GGTGAGGGGGAAATGGGGAG 77647 77666 -35 358 344 TCACAGGAGTCAGACAATAG 77114 77133 -30.2 359 345 CAGTGTTATGGTTGAAGTAG 76887 76906 -26.4 360 346 GGTCTCTGAGGGTATCTGGA 76720 76739 -30 361 347 CCAATACCATTCTTAACTGA 76604 76623 -27.8 362 348 TAGGGTTAAGATAATCCACG 76393 76412 -28.3 363 349 GCCAACTCGCCCAGCCCACA 76014 76033 -33.4 364 350 GAGGCTTGGGTACTGAGAAC 75882 75901 -31.4 365 351 GGTTCTCTTTGTCTGTTCTA 75521 75540 -28.2 366 352 TCCTGGAAGCTAATAATCTG 75349 75368 -31.8 367 353 TATCCCTCCCCCTAGCCCCC 75135 75154 -35 368 354 GTCTGTTTGTATCCTTCACC 74462 74481 -29.1 369 355 TTTGGCCTGAGACAATGGGG 73391 73410 -33.6 370 356 GCTCCTCGTTGTACCTCTGG 72707 72726 -34.9 371 357 GTGTGATGTGTTGCTGAGAA 71891 71910 -34.8 372 358 GGGTCTTGACTCGTTATCCA 71370 71389 -32.8 373 359 GCAGTGGCTGTTACCGTTTT 71180 71199 -29 374 360 GGTCCCCCACTCTCTTCTGG 70981 71000 -35 375 361 GGCTTGCGTATGCTTCATGA 70502 70521 -30.4 376 362 GCAGGTCTGCTGGTGTTTGC 70035 70054 -32.5 377 363 GCTGTGCTGAGAGATCCACT 69773 69792 -32.3 378 364 GGGTCACCACAACATCCGCC 69167 69186 -32.3 379 365 GATGGAATAGAAGAAGGCAG 68819 68838 -35 380 366 ATAGAAGAAGGCAGGGGGAA 68813 68832 -33.5 381 367 TTACATCAGATGACACTTGC 68594 68613 -26.9 382 368 GAAATTATTCTGATAACCCA 68340 68359 -21 383 369 ACAGTCCACTGGATGATGTT 68148 68167 -28.4 384 370 GTCTCCTCCAGGTTTCAGTT 68009 68028 -31.6 385 371 GGGGTTTGTATTTCTGGCCA 67747 67766 -32.3 386 372 TTATGGGCAGGGCTTGGTGG 67614 67633 -35 387 373 TTGGGAAGCTGAGGAGGTGG 67571 67590 -35 388 374 AGAAATCAAAACTGAGACCA 67219 67238 -27.6 389 375 TTCACTGCTCACAACTACCA 66887 66906 -28.6 390 376 ACCAGTATGTCCATTCTTTC 66806 66825 -28 391 377 CTTATTAGTCGTGCTAGCGG 64759 64778 -33.7 392 378 CAAGGGCTGGAGAGGATGTG 64172 64191 -35 393 379 GAGCCACTGCATTCAACCTC 62925 62944 -32.9 394 380 CAGGCACTTCACTAGGTATT 62852 62871 -29.1 395 381 GACAAAACCCAGTTGGCTTG 62586 62605 -30 396 382 ACGCGTTCTAAGCCAGAGGG 62510 62529 -33.4 397 383 GATGGGATATCAAGTAGGCA 62100 62119 -31.6 398 384 GGGCTAGACATAAAGTACAG 61908 61927 -29.9 399 385 GCCACTGTAGTTGCTGCTGT 61751 61770 -27.8 400 386 AAGATGGGAGGGAGTAGAGA 61706 61725 -35 401 387 CATTGTCATCCTTTAACCTC 61375 61394 -25.9 402 388 TTCTGCAGACTGGGGCAGGG 61042 61061 -35 403 389 GAGTTCTCACCATATTGCCC 60894 60913 -32.5 404 390 TCAACGCTCAGCTCTTGTCC 60722 60741 -29.9 405 391 TTTGGCCAGCGGGGAGAGGG 60158 60177 -32.2 406 392 CTTAGGGTAGAGTGCAGTGG 60125 60144 -30.6 407 393 CTGATCCTCTATTTTGTTCC 59511 59530 -29.9 408 394 CAGACACTACCCCCACCCCA 59002 59021 -34 409 395 CTGTACGATGCTTCGGAGTG 58612 58631 -29.4 410 396 GGATCCATTTGGCTATGTTA 58387 58406 -31.1 411 397 CACAGGAGGAGATAGGAGGT 58207 58226 -28.9 412 398 AACCCACAATCAGCAGGCCT 57935 57954 -32 413 399 AATTTTCTCCCAAAACCCAT 57513 57532 -25.8 414 400 GCTTTGCATGGAAATACAAA 57466 57485 -28.1 415 401 GGAAAACTGCCATGTGTCTT 57280 57299 -25.7 416 402 GAGGGAGGACTGCGTGATCC 57035 57054 -32.2 417 403 ATAGCAGGACAGATGGACGC 56988 57007 -31.9 418 404 GGCCAGGCAAAGTGGCTCCC 56800 56819 -33.4 419 405 TGTACAAATACAGCAAAGCT 56329 56348 -26.5 420 406 GCACAATGTCCTTCTTTCCA 56208 56227 -30.3 421 407 ACATGTAAGGCAATGTGCAG 56034 56053 -30 422 408 ATGAGAATTCTGATGGAGGA 55891 55910 -30.5 423 409 CTGGAAGTAGTCAACAATGT 55747 55766 -29.1 424 410 GAGACACCGAGAGACAGAGA 55599 55618 -35 425 411 GAGGGACGAAGAGGCCAGGG 55466 55485 -34.3 426 412 GCAACCCAGAAGACAGCCCT 55214 55233 -31.6 427 413 GGAATACATCGCAGGAGAGA 55013 55032 -35 428 414 GAGTAAGGGAGAGGCATATG 54848 54867 -35 429 415 CATTTCCTTCTCCCTCAGCC 54549 54568 -35 430 416 GATCCATTAGAGTTACATTA 54264 54283 -26.9 431 417 ATCTCGTGATTTTTGAAAGC 54044 54063 -28 432 418 AGCAAAGGACTTACAAGACA 53854 53873 -28.9 433 419 GGAGAGCAATCAAGTATAGT 53809 53828 -33.9 434 420 GGCAAAAATGGACAGACCAC 53253 53272 -31.1 435 421 GTGCAACGTTTGTCTAGGGG 53155 53174 -33.2 436 422 GAGTAAGGTGGAGGGAAAAG 53060 53079 -35 437 423 ATGGGGTGGGTGAGGCAGAG 52870 52889 -35 438 424 CCTTGCTCTACTAGTCCTCA 52550 52569 -35 439 425 GGCTGGTTGGATGTCTTCTT 51925 51944 -29.6 440 426 AAGACCTACCTAGACATCCA 51578 51597 -29 441 427 GAACTACCACGGGAGTCACT 51379 51398 -29.7 442 428 GAGGTCAGATTCTAGGGGAC 51286 51305 -34.6 443 429 GCAGAGAAAACAGACAACTG 51250 51269 -27.8 444 430 CAGAATCTGAGGTGGAGGAG 51122 51141 -32.2 445 431 GGTGAAGGCAAGTAAGGGAA 50907 50926 -35 446 432 GCACATGTCTGTGGTGATGC 50685 50704 -33.3 447 433 GCAGATCCTTCAGGAGGTAT 50163 50182 -32.8 448 434 GTGGAGATGGAAGATAGTGG 50061 50080 -34.2 449 435 GCTGTACAATGTTTGTGTCC 49907 49926 -27.3 450 436 GATGCAGTCTCAGTCTATCG 49569 49588 -32 451 437 GCCCGGCCTACTTTTTATCT 49298 49317 -31.4 452 438 AGAGCCCCATCATTCACTGG 49030 49049 -31.2 453 439 GGGAACGTAGTGATGGGACA 48743 48762 -31.6 454 440 ACGCAAAGAAGGAGAGTGGG 48676 48695 -35 455 441 GGAGGCTATAATCACAGGGC 48635 48654 -31.2 456 442 TAGTGAGAGAATGGTTTGGG 48415 48434 -32.4 457 443 GGGCTGGCCCAGATCTGCCT 47887 47906 -28.7 458 444 CTCACTCTCCTGGCAAACTC 47757 47776 -29.8 459 445 GCAGAGTTAGCTACTGAGTT 47670 47689 -29.2 460 446 GCATACTACATGGGTGAACC 47282 47301 -33.5 461 447 GGGGAGATGAAATGTTCTGG 47137 47156 -30.4 462 448 AGAAGAGTAGATTTTATGGA 47018 47037 -28 463 449 TTAGGTTAGGCAAAGAGTTC 46545 46564 -27.5 464 450 GGCCACGGCCGGGCGCGGTG 46202 46221 -28.5 465 451 TCAGCAATTCCTCTCACAGG 45725 45744 -30.4 466 452 GACACAATGGGGCACCTACT 45490 45509 -31.3 467 453 GATCAGGGATAGGGAGTGAG 45408 45427 -35 468 454 GAGGTGTGCATAAGCAGGTC 44640 44659 -29.8 469 455 TCAAGTGATGGGCAGGGGAG 44427 44446 -30.3 470 456 CTATGAAGGCAGGCTGGAGC 44314 44333 -32.3 471 457 GGACCTGAAGACTATATGGG 44051 44070 -33 472 458 GGTCTGAGGAGAATGGCTGG 43518 43537 -35 473 459 GCAGAGAAGCCAGGGAAGAT 43347 43366 -33.4 474 460 GACAGACTACTAGAGAGGAA 43320 43339 -33.5 475 461 TTCAGTGGAGGCAGTGGGGA 42829 42848 -34.4 476 462 CAGTGAGGAATAGTGTGACC 42732 42751 -35 477 463 TCAGGTAGAAGAAGTGAGGC 42611 42630 -33.6 478 464 GACACAGTATCCCGGGCAAG 42407 42426 -31.2 479 465 GCAGAATACACTTAATGAGG 42370 42389 -31.7 480 466 AGCATATGTGAGGTGGGTGG 42228 42247 -35 481 467 TTGGGGGCCCAGCTGAGTGG 42102 42121 -35 482 468 GACTAGCTGAGGAAATGTGG 41916 41935 -32.2 483 469 GGAGCTGATAGGAGAAAAGG 41889 41908 -33.3 484 470 GGTAGAGAATAAGAGAAGGA 41698 41717 -33.6 485 471 GTGGGCAACCATCTGGTACA 41575 41594 -31.7 486 472 AGTTGGGTAGTGTGTGGCAA 41420 41439 -34.5 487 473 AGTGGGGACACAAGGAAGGA 41311 41330 -35 488 474 AAGCTCACTTCATAGAGGGG 41218 41237 -32.3 489 475 AAGTTCTGATTCCCTTTACC 40972 40991 -28.6 490 476 TCTTCTTTTCACCTGTAGCC 40681 40700 -27.8 491 477 GAACCACAAGCTATTTTAAG 40373 40392 -28.2 492 478 CCCAGATGTTATTTATAGTG 40347 40366 -30.2 493 479 CTGGTTCTTGGTGGGGAGGG 40116 40135 -35 494 480 GGCTAGCCTTATCTGGATCC 39931 39950 -29.2 495 481 AGATGCTGGTGAGGCCGTGG 39384 39403 -35 496 482 ACATGGAGGCGGCTGGAAGC 39042 39061 -33.4 497 483 GAGGGAAAAAGCATGGGTGG 38888 38907 -35 498 484 AGGCTCCTTGGGGGAATATA 38702 38721 -30.2 499 485 AGAAGGGAAGGAGGGAAGGA 38110 38129 -34.1 500 486 TGACAGCAAAGTATATGGTC 37834 37853 -25.2 501 487 GGGCATACAAATTGGAAAGG 37681 37700 -32.3 502 488 CACTACCCAAAGTAATCTGC 37303 37322 -30.9 503 489 GCTGCCAAGAACATACATTT 36999 37018 -31.8 504 490 AGAATGTATAAGGAGCTCGA 36613 36632 -32.2 505 491 GTATGACCACTACGGAGAAC 36323 36342 -32 506 492 GGCAGGGGGAGTGAGGATGG 35953 35972 -35 507 493 GGTCCCTTCTCACATTGCTG 35610 35629 -29.6 508 494 AGGCGAAGAAGGAGGCACCT 35451 35470 -35 509 495 TGGATTAGGGGTGTCAGGCC 34410 34429 -31.5 510 496 GAACCAGAACTACACATGCA 34264 34283 -27.2 511 497 AACTCCTCACTCAGATATCA 34181 34200 -28.1 512 498 GAGGTTGAGACAGGAGGACT 33999 34018 -35 513 499 GGCAAACCCTGAGGATATGT 33538 33557 -26.5 514 500 CTGATATTGGCTTCTGTTCC 33326 33345 -28.4 515 501 GTTCTCCATCTCCCTTTTCA 33098 33117 -29.1 516 502 CTTCTTCTCATGCTCTGCTG 33048 33067 -30 517 503 AAAGGGTCTTGCTATGTGGC 32753 32772 -31.6 518 504 TACAGATATATGAGAGGGGA 32418 32437 -29.8 519 505 GGTGTAATTCTCAGTCTGAG 32263 32282 -30.4 520 506 TATCACTGCAACTCTACTGC 31899 31918 -27.3 521 507 GCAGGTAGGACCATCAGTGA 31507 31526 -31.4 522 508 AATACCATAGGAAGAAGCTG 31386 31405 -29 523 509 GCTAATCTCAGTTCCAAAGA 31194 31213 -23.8 524 510 GGGATAGTCAGCTGCTTGGC 30962 30981 -29.8 525 511 GGCCTATGTGGTTTAACTTT 30655 30674 -28.1 526 512 GGCCACCATGGTGAAGCCCC 30363 30382 -31.3 527 513 TCCAGCGTGGGCGAGAGAGC 30216 30235 -35 528 514 CTGGATTATGATCAATACAC 29985 30004 -25.3 529 515 AAGTATCTAAAATCTTGGCA 29665 29684 -24.1 530 516 GCACAACTGTCAACCCAGGT 29474 29493 -28.3 531 517 GGGACCGGGAGCCTTTAAGA 29317 29336 -32.7 532 518 GTACATGACTACGAGAAAAT 29135 29154 -25 533 519 GGTCTGGTCCAGAGTAAGAG 29017 29036 -31 534 520 TAGCTGGTTCATGAAATAGA 28634 28653 -27.8 535 521 GTCTTTCAGTGGGTTTGTAT 28587 28606 -29.8 536 522 TCCTCACACCCAACATGCCA 28417 28436 -30.8 537 523 CCTCAATTACTTGTCTATAA 28292 28311 -25.1 538 524 CCCTCCCCACTCCCCACAAC 27903 27922 -35 539 525 ACTCTTAAGGCAGAACATGA 27622 27641 -27.4 540 526 AGCGTTCCTCTCCCTCTCCA 27310 27329 -35 541 527 TCCATCCCTTCTAGTCTCCT 27275 27294 -35 542 528 GTTCCATTCCACCTACGCAC 27119 27138 -30.5 543 529 AGGGGCTCGATGTGGGGGAG 26747 26766 -35 544 530 CCATGCCTACTCTTCCCTCC 26401 26420 -35 545 531 CTTCTTCTCCTTCACATCCC 26296 26315 -35 546 532 TCTTCCCACTCTGTCCCATT 26142 26161 -35 547 533 ACCCTTCCTGATGCTATTCC 25928 25947 -31.6 548 534 GCTCCTAGATCAGGGATTCT 25620 25639 -28 549 535 TGCAATCTGCTGACCCACAC 25595 25614 -29.8 550 536 GATCCACTGCTAGGTCTTGT 25325 25344 -25.7 551 537 TTACCAGGTTGCTAGAGGGG 25153 25172 -34.3 552 538 AAAAGAGGGAGCAGGGGGAG 25032 25051 -35 553 539 TTGAAAGTCACACACCTATC 24507 24526 -31.6 554 540 GTCACACACCTATCTTTCAT 24501 24520 -28.1 555 541 CTCTAACTGCTATGGTATCT 24080 24099 -29.9 556 542 GCTTCGGTATTAAGGTATAT 23972 23991 -26.9 557 543 TAAATAAGATATGAGACAGC 23791 23810 -26.5 558 544 GAATAATCTTTCCTCTGAGC 23669 23688 -26.3 559 545 CCTTGCCCAGCCCTGTAGAG 23166 23185 -32.5 560 546 CGTCACTGGTGTACTTCTGC 22800 22819 -34.6 561 547 CAGCATCTGTCCACCGAAAG 22580 22599 -28.5 562 548 TCCACTGGGCTTGCTTCCAC 22343 22362 -26.4 563 549 GGGAGAGTAACTAGAAAGGC 22215 22234 -34.8 564 550 TCTGGGAGGATGAGGTGGGA 21995 22014 -35 565 551 AGCAGGGCACGGTGGTGCAC 21889 21908 -30.7 566 552 GATGACCAGTGACAATGAAG 21656 21675 -30.2 567 553 GAGAAGAAAATAGGTGCCCA 21503 21522 -30 568 554 TATCATGACTGCAACAGACT 21441 21460 -26.4 569 555 GCTAGTCCTCCAGGTGATTC 21119 21138 -35 570 556 GGCAGGATCAGGTTGACACC 20995 21014 -33.4 571 557 GACCCAAATCCAAAATATGG 19806 19825 -25 572 558 ATGATAAGATGTGGCCGGGC 19596 19615 -33.8 573 559 GATGTGGCCGGGCATGGTGG 19589 19608 -35 574 560 TTAGGGATGGGTGCAGGGCT 19067 19086 -32.5 575 561 AAGCAAGGGACACTGACACA 18888 18907 -28.8 576 562 GCACTTTGCAGATACCACTT 18541 18560 -30.5 577 563 GGCAACTCTGTGTTGGTTCC 18467 18486 -30.6 578 564 CAGAAGTGCTACCCCAGTGA 17973 17992 -32.6 579 565 ACAGCCCTTAAGAATGATGC 17458 17477 -28.4 580 566 GCAGCCTACGGATCAAGGAG 17201 17220 -34.2 581 567 CAGGCATCGTGGCAAACACC 16972 16991 -31.3 582 568 GCTGAGACGGGGGGATCTCT 16670 16689 -34.2 583 569 AGGGAGGGAGGCAAAGAGGG 16369 16388 -35 584 570 GGCCTAGAATTGAACATGCA 15860 15879 -28.5 585 571 GAATGAGCTCTTGGAAACTA 15662 15681 -27.9 586 572 GGAGACTGTCCCTGGAGGAT 15492 15511 -27.3 587 573 TAGCAGACTGCCAACCAAGC 15157 15176 -28.1 588 574 CAGACTGGAGCCAGAGAGGG 14868 14887 -35 589 575 GGAGGAAACAATATGAACTG 14819 14838 -27.7 590 576 GCACTGAGCCATGACCACGC 14221 14240 -35 591 577 GGGTTTGAGGCACGGTGGGA 14062 14081 -35 592 578 AACAAGTAGGCCGGGCACAG 13717 13736 -35 593 579 GGCTGAAGCAGGAAGATAGC 13215 13234 -28.9 594 580 TCTGGAAAATGAGGCCACCC 12971 12990 -30.6 595 581 CCTTCTTTGAGCTGGATACC 12740 12759 -30.6 596 582 AGTATTCCAAGCAGTTTGCA 12546 12565 -29 597 583 GGGGGATTAGAGTGCTATAG 12428 12447 -33.7 598 584 TCTTGCCCACGTGATGCCTT 12218 12237 -35 599 585 GGGAGACTGACCTCACTACT 11919 11938 -32.9 600 586 GAGAATGAAGCCTGAGGACT 11655 11674 -32.6 601 587 GCAGTACTGGGGCATGGATC 11549 11568 -31.2 602 588 CTTAATTTCTCTGTGCCTGT 11368 11387 -28.5 603 589 TAGAAGAACAGTGTGGGGCC 11169 11188 -31.9 604 590 AAGAGAATGGTGGTCCCAGA 10762 10781 -32.2 605 591 CAAGAGAGAGTAGATTAGGA 10728 10747 -31.5 606 592 CCAAGGCCAGGTGCAGTAGC 10599 10618 -30.5 607 593 TATTATCTGCGGCAATATGG 9878 9897 -24.7 608 594 TTACAAGAGGCTGGGACGTG 9739 9758 -35 609 595 ATCCTCCCTACCAAAAAACC 9397 9416 -34.2 610 596 ACACATCCCTGGCTTTTCCC 9225 9244 -32 611 597 CTCCGTCCTTTCCACTTCTC 9137 9156 -34.7 612 598 GTGTTCTGACAGAAAATGGG 8767 8786 -35 613 599 TACACAACCCCTTACGTGAA 8431 8450 -32.6 614 600 CAGGGAGCAGTACAGCTTGG 8144 8163 -33.3 615 601 GCCAGAAGAAAGCAAGAAGT 8054 8073 -32.5 616 602 AGCAAGGAGTAACCAAATAG 7801 7820 -29.6 617 603 GGGGCAACCAGGTAGGTAAC 7596 7615 -33.7 618 604 TGATGAATAGGAAGTACACA 7574 7593 -30 619 605 ACATTTAAGTGGGGGAGGGC 7334 7353 -35 620 606 GGGGGAGGGCTGTTATAGAA 7324 7343 -33.5 621 607 TCTGGGCCCAAACTAAGTGG 7027 7046 -32.5 622 608 TTTGTCTGCTGTTCATGATG 6852 6871 -29.9 623 609 TTGTCTGCTGTTCATGATGA 6851 6870 -31 624 610 GACAAGACGCCACAGTGGAA 6338 6357 -32.9 625 611 GGCTAGACTAGTGCAGTGGC 5910 5929 -35 626 612 CCTCCCGCCATGGGCCTCCC 5707 5726 -29.7 627 613 GTGAAGACAAGGGAAGGGGG 5385 5404 -34.5 628 614 GCCTCTTAGGGACTGAGCTA 5217 5236 -31.3 629 615 AGTCTACTCAAAAACCTGGG 5067 5086 -28.2 630 616 AGGCCAAGGTGGGAGGATAC 4891 4910 -35 631 617 GCACATGACTCTGCAACCCC 4544 4563 -29.4 632 618 GCCCAGGAGGTTGACAGTGT 4263 4282 -33.1 633 619 ACGGGTGAGGAGGAGTTAGG 4125 4144 -35 634 620 TTTGGGTGATGGAAGTGTTC 4081 4100 -31.4 635 621 GCTCTATCTCGTAAGATTGC 3732 3751 -22.9 636 622 ACCTGTTTCCTATTGTCGCT 3486 3505 -30.2 637 623 CAGGGGAAAAGACAGATACG 3352 3371 -29.5 638 624 GAGACCTGCATTAGCAAGAG 3284 3303 -29 639 625 GGGCTCTTTGCTATCCTACC 3135 3154 -29.1 640 626 TAAGACTCCTTTTCAGTAAC 2867 2886 -29.8 641 627 AACATTAAGGTCGCTCGGTC 2741 2760 -35 642 628 CAACTGGTCACGGGGAGGAG 2566 2585 -35 643 629 TCGCGCGTGGCTGGGGGCAG 2418 2437 -35 644 630 AACCGACGGGAGGAGGGGGA 2388 2407 -35 645 631 AGCGCCCTACCTGAACCCGC 2162 2181 -24.4 646 632 GGGCGCGCCCAGCCCGGTCC 2117 2136 -26.4 647 633 GATGCTCACCGATACTGATA 54427 54446 -24.2 648 634 CTCCCTCGTCTTTCACAGTC 54363 54382 -26.4 649 635 CTGGCTTGGATCAAACTCTG 54322 54341 -24 650 636 GACATCAATGATCTTACCTG 68095 68114 -25.7 651 637 GATGAAGTCACAGAACTATC 68044 68063 -22.1 652 638 GGAAGAACTGTCTCCTCCAG 68018 68037 -25.2 653 639 CCTCTCACCGAATCTGATGA 79673 79692 -25.9 654 640 GAGCCAAACATCATCTCTCC 79617 79636 -25.3 655 641 CAAACATCATCTCTCCAAGC 79613 79632 -27.6 656 642 GCACTTACGTATTGAGACTC 80004 80023 -23.3 657 643 TGCCAGTCCGAGCAGTAAAC 79971 79990 -26.8 658 644 GCCAGTCCGAGCAGTAAACA 79970 79989 -28.3 659 645 ATCAAAGGAACCATTTACCT 82435 82454 -23.4 660 646 GTTATTATCAGCCTTTAATG 82386 82405 -24.6 661 647 ATCTTGTAGCCTATGGAGGG 82335 82354 -31.3 662 648 GATCAGGCGGAGCGGACCCT 88382 88401 -18.8 663 649 ACCGTGCTATGCCACTGTCT 89778 89797 -24.4 664 650 TACCATCTAGGAAATACCCC 92574 92593 -28.2 665 651 GCGTCGCCAACGTCGCTGGT 92530 92549 -24.1 666 652 CTGGTAGCTGCTGGCACAAC 92515 92534 -21.7 667 653 TGGAGTGATCAGCAAGCTGC 92464 92483 -29 668 654 GTTCATGTGGCTTTCAAAGA 94893 94912 -26.9 669 655 GGTTAGGGCCACAGCTTTCA 94765 94784 -26.3 670 656 AAAGCTGGGCAATATGTACC 100084 100103 -26 671 657 TCTCTGACTGGCATCAGCTG 100021 100040 -27.3 672 658 GAAACGATAGCAAAGGTGGT 119909 119928 -31.4 673 659 ATCATTGTAAGCCAGACAGG 119869 119888 -29.9 674 660 TCGTGGCATCGTGTAAAGAT 119840 119859 -27.6 675 661 GTAAAGATTACAAATTGTTG 119828 119847 -24.5 676 662 AGTGGGTTATATGGATGAGT 121265 121284 -24.8 677 663 CCACACTCTGAGAGGAATGC 121231 121250 -26.3 678 664 CTGAGAGGAATGCTTTTCTA 121224 121243 -25.8 679 665 GAAAACTTCCTACTTACCCA 127494 127513 -20.4 680 666 CAATCTTCTTGCTCTCCTCC 127447 127466 -25.4 681 667 CCTCCATAATAACTGCTAAC 127432 127451 -26.8 682 668 TCATATCATGACCTGTATCT 127404 127423 -26.6 683 669 CTATCCCCAAGGGCACTGTC 127375 127394 -27.3 684 670 GTTATTTTTTGTACACAATA 127172 127191 -24.6 685 671 TTTTGTACACAATATTTTTG 127166 127185 -21.6 686 672 GGAACATCATCAATAGTCCT 127096 127115 -25.9 687 673 CAATAGTCCTGGTTTCGATT 127086 127105 -25.7 688 674 TTCGTCGAATAAGCTCATGC 127049 127068 -24 689 675 CAGGGAAGTTCACAGGGCTC 127006 127025 -28.2 690 676 GGCTCTTCAGAAACCATGTT 126991 127010 -24.4 691 677 GGTCCTTGGTTGTTTGTTTA 126948 126967 -26.4 692 678 GGTTTGGTGTGATGTCTGGT 126886 126905 -28.5 693 679 GATCCACCACTGCCTGACTT 126861 126880 -29.1 694 680 CTGCCTGACTTCGAAGCTCA 126852 126871 -25.3 695 681 TCAGGTGACATAGAATCCCC 126823 126842 -26.1 696 682 GGAAAGTAACCTTTGTCTGG 126798 126817 -22.3 697 683 GTGGATCTCATTGCTTTGCC 126712 126731 -26.7 698 684 CCAGCAACTTCTCACTCTGC 126675 126694 -22.4 699 685 TTGTCTACTGGAACAGATCC 126610 126629 -23.2 700 686 CTCTAACTTGGCGTCAAAGC 126575 126594 -21.2 701 687 TCTCTGTACTGTTTAACATC 126541 126560 -23.5 702 688 CTTGGCAGTCAGAAGGAGAA 126507 126526 -26 703 689 CACTCATCTGAAATTCCTAG 126478 126497 -18.9 704 690 ACAGTTACAATTGGGAGTTC 126368 126387 -22.8 705 691 CTCTTTAAAGAGCAAACTGC 126335 126354 -24.3 706 692 GCATTGTGACAAGGACATAC 126303 126322 -29.5 707 693 TCACCTTTCTCTAAAGTGGT 126268 126287 -29.1 708 694 AGTAATTACTGTGCCTGGCA 126248 126267 -29.6 709 695 AGTCCTTGCTAACCGTACGG 126104 126123 -32.4 710 696 GCGCCATACAAGTCTCCTGT 126073 126092 -30 711 697 GGTCACTCACCTGCACAGCA 128529 128548 -29 712 698 AGTCAGGCACATAAGATGAG 128445 128464 -27 713 699 AGCCACCCAGCAAGGACCTC 128421 128440 -31.1 714 700 GCTGAAGTGTGGAATGAAGC 147151 147170 -24.8 715 701 GTGGAATGAAGCAGATTGGA 147143 147162 -26 716 702 ACTCGTCTCTGGCTACTGGC 147077 147096 -30.9 717 703 ACAGTCCATTTATCCATGTC 147050 147069 -29.6 718 704 GACATGAACACGCATCTGCC 151869 151888 -27.1 719 705 TCAGGTATTCAGACAGCATT 151846 151865 -32.8 720 706 GCTCCTGCAACCGGTCTTCA 151810 151829 -27.1 721 707 GCAACCGGTCTTCAAGATGC 151804 151823 -25 722 708 CCCTAGAACTGGGAGGTTAT 157234 157253 -24.6 723 709 GTATACAGAACAAGAAACAG 157187 157206 -24.9 724 710 ACATATCCAACATCTAAGTA 157161 157180 -19.6 725 711 GACATCTACCAGAAGTAGGC 157113 157132 -24.2 726 712 TTTAAGTGATCATCCCCAGA 157074 157093 -30.4 727 713 ATAAAAGCCAGAAATATTTA 156969 156988 -18.2 728 714 TCTATAATGTAAAAGATCCC 156904 156923 -23.9 729 715 GTAACAACTGTCTGTACCAT 156843 156862 -27.4 730 716 CAACTGTCTGTACCATAACT 156839 156858 -25.9 731 717 CTTTTCACATTCATCATAAT 156789 156808 -21 732 718 CATTCCTTGGGCTTTGCGTA 156743 156762 -23 733 719 AAGAATGACTATGGTGAAGG 156719 156738 -26.3 734 720 GAATGACTATGGTGAAGGTA 156717 156736 -27.8 735 721 GAAGGATTAGGAATTACAAA 156685 156704 -27.2 736 722 GGGGAGGAACAAAACAGTAA 156645 156664 -22.8 737 723 CCTTCCCAGCAAAGCTTAGC 156574 156593 -21.8 738 724 GCTTTAGGAACAACAATAGC 156556 156575 -25 739 725 AGGAACAACAATAGCATGAG 156551 156570 -25.6 740 726 GGACTAGAATTTCTAAACGG 156516 156535 -24.4 741 727 CTAAACGGTAATTAAACATT 156504 156523 -15.5 742 728 GGTTAAGGAGACACCCAGAC 156463 156482 -25.7 743 729 TTCCAAAATCAGACAACTGG 156021 156040 -22.2 744 730 CAAAATCAGACAACTGGATA 156018 156037 -24.6 745 731 AGGTAATAACTGGGACTAGG 155910 155929 -26.8 746 732 GGGCCAAACAGATTGCAGAC 155879 155898 -24.2 747 733 CAATTTCAGGTTTACATTCC 155855 155874 -24.6 748 734 ACAGCAAGGCAAAATGTGAC 155783 155802 -24.4 749 735 CAAGGTCAAAGACAACTTTC 155748 155767 -22.4 750 736 AAACAGCAAGAATGGATGTG 155709 155728 -23.9 751 737 GAATAAACAAGTGGTTTTCT 155663 155682 -18.3 752 738 GTCAATGAAAGCAGAAACCA 155609 155628 -23.9 753 739 ACCAACTTTACTACTAGCAT 155593 155612 -22.9 754 740 GACCAGATAAGCAAAGAATA 155560 155579 -23.7 755 741 GTAGTACTCAAGTAAACTGA 155531 155550 -23.8 756 742 GAGGTAGTAGAAAGTATTGA 155513 155532 -26.6 757 743 ATTTACATTACTTACAAGAG 155470 155489 -24.2 758 744 TACAAGCAACACTCTATAGT 155427 155446 -23.5 759 745 TAAGAGTTATGAATAGTGTA 155317 155336 -25.7 760 746 AAGAGTTATGAATAGTGTAA 155316 155335 -25.2 761 747 TGAGGATTATTAAGCAGTGA 155294 155313 -26.3 762 748 GGGCCTTGGCTACCTGAGCA 155225 155244 -29.1 763 749 GCCGATGCTTTGGTGCAATT 155179 155198 -22.1 764 750 CATTTATGTTAAATTAGTGA 155144 155163 -23.3 765 751 GAATATGTAAATATTAATAT 155126 155145 -15.9 766 752 TTATAAGAACAGTTCATACT 155106 155125 -18.1 767 753 AGTAGTATTCTCATTTTGGG 155068 155087 -24.2 768 754 CTTAAAAAAAAGGTTGGCTT 155039 155058 -24.8 769 755 TAAAGGCATGTTTACCAGTC 154927 154946 -22.9 770 756 GGCATGTTTACCAGTCTTGT 154923 154942 -23.9 771 757 GGATAACATAACACATTTAC 154852 154871 -24.6 772 758 TCTTATAATTAACAGGCTGT 154815 154834 -24.8 773 759 GGCTGTTTAAAATACTTACT 154801 154820 -25.3 774 760 GAGAAATAACCAGACATTAG 154779 154798 -21.2 775 761 ATAGATCTCTGTACCTTAGA 154720 154739 -22.1 776 762 CTCTGTACCTTAGAAATTAA 154714 154733 -21.4 777 763 GAGCTGTGACAACACTGCAG 154630 154649 -18.9 778 764 GTTGCTTTAATTTCATTTGT 154526 154545 -22.3 779 765 CACCACCCAAAAGTCCAGTC 154494 154513 -29.1 780 766 GCAGAATACCCTGGTGACTG 154430 154449 -28.1 781 767 TAAATGCATGTTGTGTCTGC 154369 154388 -26.9 782 768 TTCTACCTATTTTCCCACCA 154340 154359 -21.9 783 769 GGAGTATTTGTGCAACATAT 154292 154311 -27.5 784 770 GAGTGCTTGCTACAGCAGCC 154265 154284 -30.7 785 771 GTGGAAGATCACTGGATTCA 154241 154260 -25.4 786

TABLE 2 Example antisense oligonucleotide modulators to downregulate FNIP1 Target start  Target stop site with site with reference to  reference to SEQ ID SEQ ID SEQ ID GI ID# Sequence NO: 11 NO: 11 NO GiTx-45 CTCAG GTATTCAGAC AGCAT 151847 151866  43 GiTx-46 ACGGG AGAGCCAATA GCGCC 126088 126107 148 GiTx-47 GCTCT CTTCCAGGCA TGTCC  89745  89764 304 GiTx-48 GTCTC CTCCAGGTTT CAGTT  68009  68028 385 GiTx-49 TGGCA TCAGAAGAGC ACCGA  79589  79608 349 GiTx-50 TGGCA TCAGCTGATC TCCGG 100013 100032 259 GiTx-51 TTCTCTTCTTACCCGCAGCC 150171 150190  49 GiTx-52 CGCAGTGAAAGGGACATAGG 144307 144326  70 GiTx-53 GATGTCCTGTCCCTTTCCTC 123802 123821 155 GiTx-54 TGATCCGAGGTTGGCTGATT 115276 115295 191 GiTx-55 AGAATGGTGGTTACAAGAGG  92919  92938 287 GiTx-56 CAGTGAGGAATAGTGTGACC  42732  42751 477 GiTx-57 AACAAGTAGGCCGGGCACAG  13717  13736 593

TABLE 3 Example antisense oligonucleotide modulators to upregulate FNIP1 Target start Target stop site with site with reference to  reference to SEQ ID SEQ ID SEQ ID GI ID# Sequence NO: 11 NO: 11 NO GiTx-58 CAGCTATGTACATATCCAAC 157170 157189 16 GiTx-59 GGAAGCAGTGGGGAGGAACA 156654 156673 19 GiTx-60 TTGATGGTTAAGGAGACACC 156468 156487 20 GiTx-61 ACTGGGACTAGGTAAAAGGT 155902 155921 22 GiTx-62 GTCTCTTAGACCAGATAAGC 155568 155587 23 GiTx-63 CTATGGGTTCATGAGGGTTG 155258 155277 26 GiTx-64 GGGTGGATAACATAACACAT 154856 154875 28 GiTx-65 GCATGTTGTGTCTGCTTCCT 154364 154383 30

TABLE 4 Example antisense oligonucleotide modulators targeting SEQ ID NO: 1 Target start Target stop site with site with reference to reference to SEQ ID SEQ ID Binding SEQ ID No ASO sequence NO: 1 NO: 1 Score NO 1 CCGCTGCGACTGAGTAACTC 2066 2085 -29.8 787 2 GTCGGGGCCATGATGCCGCC 2205 2224 -25.5 788 3 AGGGTCGGGGCCATGATGCC 2208 2227 -28 789 4 CCCGCGCCAGAATTGCCCCC 2335 2354 -28.3 790 5 CCGGCGAGCACCGAAGCCAA 2362 2381 -29.3 791 6 GGACCAGGTGCCGGCGATAG 2431 2450 -14.2 792 7 CACTCGGATCAGGCGCGTCC 2466 2485 -16.8 793 8 GAATCTCCGGTCGCCACAGC 2600 2619 -20.4 794 9 AGAATCTCCGGTCGCCACAG 2601 2620 -17.9 795 10 CGCAGAGGTGCCCAGGCGGC 2653 2672 -20.9 796 11 GTCACGTACACAATAGGTTG 2713 2732 -27.3 797 12 TTTTTCGTGACTGTTGCTCC 2752 2771 -26 798 13 GTGTTCGAGAGATCAAATCT 2947 2966 -25.3 799 14 GGATTTTGGGCAAGCCGCCT 3120 3139 -28.5 800 15 GCGCGCGTGCACACACACAC 3558 3577 -25.6 801 16 CACCTTAGGTCCGAAGTAAT 3729 3748 -25.3 802 17 TGGATAGCGGAAATGGTGGT 3938 3957 -32.8 803 18 GTGGATAGCGGAAATGGTGG 3939 3958 -35 804 19 GTACTTGGCACGATTTTCTG 4095 4114 -27.7 805 20 AGGGGGCCCTCATCAAGTAG 4200 4219 -28.8 806 21 TGTTAGGAATACTCGATTTT 4602 4621 -19.2 807 22 GGTCAATCGTGATTCCTTTT 5127 5146 -25.7 808 23 GTCACTGGACATAGCGTGGC 5326 5345 -27.3 809 24 AGTCACTGGACATAGCGTGG 5327 5346 -26.3 810 25 ATCCGGCCCTTAGTCACTCC 5958 5977 -30.6 811 26 AATCCGGCCCTTAGTCACTC 5959 5978 -29.5 812 27 TGAAAAATGGTTCCATTTTC 6642 6661 -20.7 813 28 TCTGACACCCCGTCACCTCT 6826 6845 -22.4 814 29 GCACATGCATAATCGCTCCT 7189 7208 -25.4 815 30 TTACTTTTAATAATAGGAGC 7539 7558 -20.4 816 31 TTTCATGACGGCAAGGACAA 8113 8132 -30.6 817 32 ATTTCATGACGGCAAGGACA 8114 8133 -30.4 818 33 TGGGATGAATTCCTACTGCC 9176 9195 -15.9 819 34 GGTTAACGTCTAACTTGTCT 9305 9324 -21.8 820 35 GCTAGGATTTAGACGAACTC 9706 9725 -27.3 821 36 TTCATAACTGGATCTCTGAT 10084 10103 -23.1 822 37 CAGTAGTTCCGGCCGCCCCC 10629 10648 -30.6 823 38 CCAGTAGTTCCGGCCGCCCC 10630 10649 -29.7 824 39 GTTATGCGAACGTGATCTCT 11569 11588 -27.2 825 40 ATCATAGTTATGCGAACGTG 11575 11594 -21.1 826 41 GTAGGCGTATAAACAGGTGC 11955 11974 -28.4 827 42 TCCACTAAACAGCCGCTATC 11986 12005 -28.2 828 43 ACCTCAGACCCACAAGTAGC 13095 13114 -30.9 829 44 ACTCAGCTACACTTCATACT 13740 13759 -30.2 830 45 CTCTGGGGTTCGAGCGATTC 13946 13965 -26 831 46 CACAGAGACCGGTCCACCCA 14322 14341 -28.7 832 47 AACTTGCACAATTCGGTCCA 14375 14394 -19.4 833 48 GTCAAACACTGTGTAGTAGG 14981 15000 -25.9 834 49 TTTAAGTTAGGGTCCCTGTG 15226 15245 -22.7 835 50 GGAGCATGAAGCTGTCAGCT 15396 15415 -26.6 836 51 CTAGCAGGTCACGGCAAGCT 16135 16154 -23.5 837 52 GATGGTGAACGGACTCCTGA 16632 16651 -24.8 838 53 CCATACCGGTTAGGTCCCCA 16892 16911 -22.4 839 54 CTCCATCCATACCGGTTAGG 16898 16917 -23.7 840 55 CCACATTATTCGGGCTCAAA 17033 17052 -19.4 841 56 TGATCTGTCACTATCTCCCA 17600 17619 -21 842 57 GTCACGGACTGGTACCAGAC 17805 17824 -26.7 843 58 GCTCCTACTAACGTACTGTA 17975 17994 -29 844 59 TGGGCATGGTGGCACGTGCT 18228 18247 -34.8 845 60 GGACGAGACTGGTCATAGAT 19153 19172 -29.9 846 61 TAATCCGGACGAGACTGGTC 19159 19178 -26 847 62 TGAGGACAGGCACAATTTAT 19615 19634 -23.7 848 63 GAACTCTCATTACCATATGA 19632 19651 -25.2 849 64 ACCAGTCGTTGGCGCAGAGC 20373 20392 -23 850 65 GGCTGAGGTGTGATGCAGGA 20567 20586 -34.2 851 66 AATTGGCTGAGGTGTGATGC 20571 20590 -32 852 67 AGGGCCGCCACACGAAAGAG 20806 20825 -25.6 853 68 CAGCACGGTCGTAACTGCAA 21180 21199 -29.1 854 69 AAGGCTTCCAGGAATCAGAG 21215 21234 -32.6 855 70 GAATACAGATTCGGCATGGT 21340 21359 -25.9 856 71 GCTAAGCGGGCCTTTGCTTA 21419 21438 -24.7 857 72 GCCTTTTCTATGTGGAACCT 21692 21711 -24.9 858 73 GCTCTCGCATAAGCTTTTCC 21947 21966 -21.2 859 74 GCTGTGAAAGTAGCTTGGGA 22239 22258 -21 860 75 AGGGCAGGGTGTTATCTTAT 22632 22651 -27.5 861 76 ATTTCGCCTAGAACACATCC 22840 22859 -25.8 862 77 GAATTTCCCGCTTGACGTGT 22988 23007 -26.7 863 78 TGTGTTGGGACCGTGACCCA 23022 23041 -32.9 864 79 TTGTGTTGGGACCGTGACCC 23023 23042 -34 865 80 CTCATTACGTGGCTGGTCCC 23551 23570 -27.3 866 81 CTCCACCTCCCTTGTTACCC 24080 24099 -31.8 867 82 AGGATTCTCAGTGCAGGCTG 24578 24597 -22.6 868 83 CATTCTTATCAGACCGTCTG 24720 24739 -21.6 869 84 GAAAGCCGCTGATTACAACA 25646 25665 -23.5 870 85 CCAGCAAGACTTTGACTATA 26040 26059 -25.2 871 86 GTGACGGGTTGATAGGTGCA 27560 27579 -27.5 872 87 TAATAGCTAGGTGACGGGTT 27570 27589 -26.9 873 88 TGTAACTGGACCCAAGAATT 27866 27885 -27.2 874 89 CATTATACCACGGCTCAATG 28182 28201 -25.9 875 90 TCGAGGACATGGACCACACC 28393 28412 -31.4 876 91 AGGCGGTAACATCGGCGATG 28722 28741 -31.3 877 92 GTTCAGGCGGTAACATCGGC 28726 28745 -33 878 93 TCTAAAGTACCGCTCATTTA 29247 29266 -17.3 879 94 CCCCTTCTAAAGTACCGCTC 29252 29271 -23.5 880 95 TCCTTAACCATAAATAGACA 29755 29774 -23.8 881 96 CACCAGCGCGTAGTCACTGT 29918 29937 -28.4 882 97 CCAAAATATAGCTATAATTA 30189 30208 -24.2 883 98 CGTAATAGCCTTCGTACTGG 30374 30393 -30.1 884 99 GCCAGGACTACCGTAATAGC 30385 30404 -29.6 885 100 GATCACACCTCACTGCAGCC 30635 30654 -29.5 886 101 CCACCCTAATCGAAGCCTGG 30737 30756 -27.2 887 102 GCTATCTAAAGACTTAAAAC 31655 31674 -28.6 888 103 GATAAACGAAACAGCGGCCC 33095 33114 -31.1 889 104 GCTACCGAAAGGCTATCTGA 33185 33204 -30 890 105 TAGCCGTAGCCATTACCCTC 33338 33357 -24.9 891 106 TATCAGAACGCTTTTGAGCA 33668 33687 -20.3 892 107 GCACATCTTCATAAAATCTG 33833 33852 -24.2 893 108 TCAGATCCAGATCAAAGTTA 34000 34019 -28.3 894 109 GCACACCGGCCCCTACCGCC 34198 34217 -29.3 895 110 ATACCACGTCTGCGCCTGAC 34466 34485 -27.4 896 111 GACGAGCTCAGCGCTAAGGA 34759 34778 -23.1 897 112 GGCCATCCTCACGATACCAG 34834 34853 -32.8 898 113 GACTTCGAAGCACGGTCCCC 35166 35185 -28.2 899 114 TGCCTTCTCCTTGCACAAGA 35214 35233 -30.9 900 115 CTCCAAAACCTTCGGATGCA 35732 35751 -26.3 901 116 TCTCCAAAACCTTCGGATGC 35733 35752 -24.8 902 117 TGTAATTACACACTTAGAGA 36353 36372 -21.4 903 118 ACACTCACACTCCATTTCTC 36832 36851 -24.1 904 119 CTCTTACGATCCTAACTAGA 37382 37401 -23 905 120 TGATCATGAGTTCTTTCTTC 37782 37801 -28.1 906 121 TAATGTACGGTATCCACACA 38083 38102 -21.1 907 122 GGTGAAGAAAACATTAATGG 38212 38231 -24.3 908 123 ATGATAACGAGCAGCCATAA 38510 38529 -22.9 909 124 TCCCATTACAAACATAGACA 38931 38950 -24.6 910 125 TGTCTACTCCGGTGTGTTTT 39374 39393 -33.7 911 126 CCTGTCTACTCCGGTGTGTT 39376 39395 -34.3 912 127 ATTAACGGAAGAACGTGGAT 40000 40019 -19.8 913 128 AAAAGCATGTGACCTAACAC 40819 40838 -24.8 914 129 AATCCGAGCCAATTACATAG 41106 41125 -26.2 915 130 TAAAGCATCACTTCGAAGGT 41333 41352 -21.9 916 131 TCCAAGATCGTAGAAAGGAG 41534 41553 -23.4 917 132 TATCCTGCTTCAGCATCCCA 42352 42371 -26.1 918 133 TATGTGTGGCAAGTCCCAGA 42643 42662 -30.2 919 134 AGCGCAGAAGAACGTGTGAC 43388 43407 -20.3 920 135 CAGCATTCCGCTTCAGCACA 43875 43894 -26.3 921 136 AAATCAGGGTAGAGAAGATC 44448 44467 -25.6 922 137 CCAAGTTGTTTGGCTCATTG 44707 44726 -24 923 138 GGCTTGTTCGCTCACATGGG 44873 44892 -26.9 924 139 TCACATTCCCACGAAGGATC 45030 45049 -20.7 925 140 TGTTCTATCTACCCGGCTGG 45342 45361 -23.2 926 141 ACCATGCTACGAAGAGACAT 46047 46066 -24.9 927 142 TACCATGCTACGAAGAGACA 46048 46067 -25.2 928 143 TGGTGGTGCAGTTGTCTTGG 46282 46301 -30.3 929 144 TGAGAAGACAAGCCACAGAC 46618 46637 -30.4 930 145 TGAGCATGGCGAAGGATTCT 46740 46759 -27 931 146 TGCATGTAGGCAGGGGAAGG 47484 47503 -26.2 932 147 TACAACAGGAACACCCTATT 47943 47962 -22.8 933 148 ATGACTGCAGCACTGTTTGG 48969 48988 -27.9 934 149 GGTCTCTCGTTTGCAAGATG 49167 49186 -27 935 150 GTGTAAGGTCTCTCGTTTGC 49173 49192 -27.1 936 151 AGCCGTCAACACATTATTAT 49446 49465 -24.8 937 152 ACTTCAACTGATCCGCCTGC 49553 49572 -27.1 938 153 TAAGCATCTTAATATCAACA 50315 50334 -23 939 154 GCGGAGCACTGCACGGGATG 50946 50965 -24.8 940 155 ATAGCGGAGCACTGCACGGG 50949 50968 -20.8 941 156 TGAAGTTTGTGTGTGGCACT 51023 51042 -22.8 942 157 GCTTGGGACACGGATTAGGA 51920 51939 -30.9 943 158 GTCTATAGGGACTTATGGGG 52011 52030 -22.6 944 159 GTGGTCAGGAGTATAGTGAA 52465 52484 -24.7 945 160 GGGTGGAAGAAAAGGGAAAA 52763 52782 -27.6 946 161 GTATAACATGTTGCAGGGTG 53386 53405 -31.2 947 162 GGAGTACTTATAGGTCCTGT 54651 54670 -25.3 948 163 TTGAGCCCAGGCGTTTGTGG 55392 55411 -27.1 949 164 CGGTCAGGCACAATGGCTCA 55457 55476 -23 950 165 AGATTGTCTCATTTTTTGAT 55835 55854 -17.1 951 166 AGTCTGCGTGGAGGAAAGTA 56684 56703 -22.8 952 167 TCTTTCAACAAGCTGTGCCC 56969 56988 -30.2 953 168 TTCTTTCAACAAGCTGTGCC 56970 56989 -26.7 954 169 TTCTGGCCCTTCATAATGAA 57563 57582 -28.9 955 170 CAGACGCTTGATAAGATCAG 57683 57702 -19.5 956 171 TGATGCGCACCATAGGAACA 57843 57862 -30.1 957 172 TGGCTAGCTCTAATCCAGGA 58381 58400 -25.5 958 173 TGGAGCAAGTCGGTAGAGCC 58480 58499 -28.5 959 174 GATGATCACAGACAGATAAG 58849 58868 -25.1 960 175 TCCTGTCACAGTCCTGGTAA 58962 58981 -26.8 961 176 GAGCATACTCATGCCTGTTG 59212 59231 -22.8 962 177 GCCATGATTACTTGCTAACA 59253 59272 -22.1 963 178 AAGGGATCAAGAGCTGCTCT 59674 59693 -32 964 179 GAGAGATTTCTTGGTTTCCC 60147 60166 -22.1 965 180 GAGGGCCTAAGTGACACGAG 60626 60645 -23 966 181 CCTGAGCACCCAGCACTCCC 60707 60726 -19.7 967 182 AGGAGCTACTGTGAATTACA 61211 61230 -29.9 968 183 GGGCAGACTGGCTCGTAGGG 61640 61659 -35 969 184 GCATGATGTGAAGATCCCCC 62182 62201 -26.6 970 185 TGACTGGCAATATTTCTTCC 62457 62476 -23 971 186 TACTTATGCACGGTTGCAGA 63587 63606 -20.9 972 187 GCATACATACGCTGACGCAC 63618 63637 -19.2 973 188 TAACTGCCCGCCTTTTCAAC 64015 64034 -27.2 974 189 CTCTACTCTCACTGGCTTGT 64249 64268 -34 975 190 ATGGTTCGGTATTATTCAGC 64463 64482 -30.8 976 191 CCTATTTTCCCCCCCAAAGT 64683 64702 -27.7 977 192 GTTGACATCGGAAGCTGGTC 64869 64888 -27.5 978 193 CAGACTCACCGTATGTAGTG 64952 64971 -26.6 979 194 GCCACTTTTAAAGCCCCTTT 65260 65279 -26.4 980 195 ACGTTCAGGTAATCCCAAGG 65807 65826 -30.3 981 196 AGAGCTGACAAAAACGACGA 65911 65930 -26.7 982 197 GACTGACACCCGAGATACTG 66212 66231 -33 983 198 GGAGGTAGCTCAACCCGTGT 66303 66322 -28.8 984 199 CAAAGGGAATTAAACTTGCA 66363 66382 -21.7 985 200 TGCAAGCTATATCCGGAGAT 66515 66534 -27.8 986 201 GGTTACTTCCAGTACGACAA 66607 66626 -23.8 987 202 AGCGAAGACATTTACAAGAG 67068 67087 -19.8 988 203 GTAGGTAGGAGCGTGTCAAT 67545 67564 -28.7 989 204 GTACCTGATCGAGCAATGCC 68462 68481 -27.7 990 205 CAAGTACCTGATCGAGCAAT 68465 68484 -26.1 991 206 GGACCTTGGAGTAGTGTGGA 69273 69292 -31.2 992 207 CTCTGTTGTCCAGCCTGGAG 69783 69802 -27.2 993 208 CAGTGTTCAGAACGGGCCCC 70332 70351 -28 994 209 CCCTCAAATTCCCTGAATCT 70418 70437 -26.3 995 210 GAAATCTTAGCGAACAAATT 71062 71081 -22.1 996 211 GTGACGAAAATGGCACTTTA 71992 72011 -26.8 997 212 GGTATGGGGTAAGCATCCTG 72158 72177 -27.8 998 213 CAACATAGTGGGGCCTTGTT 72555 72574 -30.4 999 214 TTCCAAACTTTAGCTTCTGG 73695 73714 -25.5 1000 215 CCAGGTGCTCACTCCTTTGG 74301 74320 -29.2 1001 216 GCCCAGGTGCTCACTCCTTT 74303 74322 -30 1002 217 GTGGGATATCCCGTTGCTGG 74820 74839 -30.5 1003 218 GCTTCACAAAGGATTTTAAT 75409 75428 -15.7 1004 219 GGGGTACAAGTGCAGTTTTG 75663 75682 -30.7 1005 220 GCAGGGGGTATTACATCCAC 76132 76151 -21.7 1006 221 GACGCAGGGGGTATTACATC 76135 76154 -28.3 1007 222 CCTTCCCGTACCTAAGATGC 76668 76687 -26.2 1008 223 CCCATCCCCACGATACATCT 76704 76723 -27.6 1009 224 GGCTTATTTATTCTGCCGAA 77005 77024 -21.9 1010 225 TTTCAGACAGTGGTAACTAC 77557 77576 -24.9 1011 226 GTGCCTCCAGTTAACTCTGT 78289 78308 -29.3 1012 227 TACGGCCTAGCATCTACATG 78347 78366 -30.1 1013 228 GCATGCAATGCTCGAGTGCT 78609 78628 -26.3 1014 229 GCTATCTGCTAGCGGATTCT 79357 79376 -26.3 1015 230 CAGAGAGGACGATCGGTGGA 79761 79780 -28.7 1016 231 CTGCAGAGAGGACGATCGGT 79764 79783 -32.6 1017 232 CTACGAACTACACTGCCTGG 80310 80329 -27.6 1018 233 CTCTCCTGGAGCTACGAACT 80321 80340 -29.3 1019 234 GTGAGGTAACGTGATCCTGG 81597 81616 -29.4 1020 235 TTTGGTGAGGTAACGTGATC 81601 81620 -26.9 1021 236 CCTGACTTCCCGACCTGAGC 83098 83117 -25.3 1022 237 CTTTCAAGTCAAGCTGCTTG 83394 83413 -22.1 1023 238 CCTTTAAAAAGGAACACAAA 83942 83961 -22.5 1024 239 GTACACTTCGCCACTAAAAA 84172 84191 -29.7 1025 240 CCAGATACTCCCCAGTGTCT 84657 84676 -28.8 1026 241 GATGACGATGCAACACTCAG 85537 85556 -28.5 1027 242 CAGATGACGATGCAACACTC 85539 85558 -25.8 1028 243 GCGATGCCGTCTCCTGACAG 85725 85744 -24.3 1029 244 GCAGAAAAGGCTTACAACAC 86165 86184 -24.7 1030 245 AGAAAGATCCTTCTCCAAGT 86307 86326 -20.8 1031 246 ATCATTCTGAGCAAACTATC 86647 86666 -17.6 1032 247 GAACATTTTTAGTTTCTTGT 86871 86890 -21.1 1033 248 CCATCAGTCACCGGAGCCTG 87262 87281 -28.1 1034 249 GACTCTAAAGCGGCACTGCC 87417 87436 -26.1 1035 250 GAATTCCGACAAGACAGGTC 87783 87802 -32 1036 251 GCATGTGTTCGGATGAAGTC 88203 88222 -32.1 1037 252 CGCACAGCGTCCCCCCTCTC 88330 88349 -30 1038 253 AAGGGATCTTCACGGGCACA 88440 88459 -23.7 1039 254 ATTGCCTCGGTAAGTGATGG 89134 89153 -28.1 1040 255 GGGTTTCAATTGCCTCGGTA 89142 89161 -29.4 1041 256 ATCTCAATAATCTATCACTG 89353 89372 -23.5 1042 257 GTCTGATCAACCGACAGCCC 89525 89544 -27.3 1043 258 GGTCACGTAGTATCTTACTT 89900 89919 -20.8 1044 259 CAGGTCACGTAGTATCTTAC 89902 89921 -22.9 1045 260 AGTGATCCTTCCACCTCAGT 90707 90726 -21.9 1046 261 CCCTAGACTGGCGATACATT 91226 91245 -22.7 1047 262 TGAGAGCCCTAGACTGGCGA 91232 91251 -24.4 1048 263 GCCACACGGTATTATCCTAA 91863 91882 -25.5 1049 264 GCCCCACAATCACTTCTCTA 92287 92306 -27.8 1050 265 TGAGAAGAAATTTTATAACT 92529 92548 -20.9 1051 266 CCATCTTGAAGGGCGGAACC 92781 92800 -25.5 1052 267 CTGTCGGTTGAGCAATAGTG 92853 92872 -22.8 1053 268 CTTGCGATTTTGCTCCAGGT 93398 93417 -29.3 1054 269 TGAGCGTCCAGTCTGACCAC 93606 93625 -29 1055 270 GGGACAACGGCTAAGTTCAG 94067 94086 -28.1 1056 271 TGGCCATGAGATTTGCATAC 94515 94534 -25 1057 272 TACGGATGTGTTTTGGCCAG 94735 94754 -24.7 1058 273 GAGAGCCGGTATAAACTACA 94842 94861 -29.7 1059 274 GTGAGAGAGCCGGTATAAAC 94846 94865 -30.2 1060 275 TTATACATCTCACGACCAAG 95398 95417 -20.4 1061 276 TCTCTGAAAATAAGCTATTC 95605 95624 -18.7 1062 277 TCTTCGTTATTATGCTGAAC 96002 96021 -25 1063 278 GCCTTCGCCGACCAGGGCCT 96161 96180 -31.3 1064 279 GAGGGGGAACGCACAGTGGC 96452 96471 -35 1065 280 GGAGGGGGAACGCACAGTGG 96453 96472 -35 1066 281 CTAATGGGTGGCGATCACAT 96587 96606 -24.8 1067 282 TCAGTGGTCCAAATCTCTTT 96881 96900 -23.1 1068 283 TTAAATCAGTGGTCCAAATC 96886 96905 -24.4 1069 284 GTCTATGTTGTAGAATTCCC 97458 97477 -16.2 1070 285 GTTGTTATATGCTTTTTACC 98177 98196 -17.2 1071 286 TATTGCCGCAGGATATCAAC 98698 98717 -21.8 1072 287 GAGAGGCGTTTGCAAGTGAC 98836 98855 -30.7 1073 288 CTTGTGTACGGTTCCTAAGA 98938 98957 -26.7 1074 289 AACCAGAATATTGGAACAAA 99199 99218 -20.3 1075 290 GAGCCAAGGTGTCGCCACCG 99631 99650 -20.5 1076 291 GCTGGTGTGCACCTGTAGTC 100329 100348 -33.5 1077 292 CAAAATACTTAATGAGGTGG 100619 100638 -21.9 1078 293 TCACAGATATATCCGTCTTT 101034 101053 -23.6 1079 294 GTCACAGATATATCCGTCTT 101035 101054 -27.3 1080 295 CACTACGGTGCGAGTCAGTC 101136 101155 -23.9 1081 296 GTATTCGCTGGACTAAGTCC 101170 101189 -25.1 1082 297 TCCTCACCGTAATGACCACA 101338 101357 -28.1 1083 298 GGGGGTACAAGAGCGGGCTC 101360 101379 -31.1 1084 299 ACTGACCGGTCTGGCAGTCT 101714 101733 -27.5 1085 300 GCTCCTAGAAACCTGCGGGT 101916 101935 -26.5 1086 301 GGACACACCTGGGGGCAACA 102193 102212 -28.9 1087 302 TCCCAGGGCGCTATCTGAGC 102312 102331 -27.7 1088 303 GAAGGACAGGTGGGAAACCG 102585 102604 -26.3 1089 304 AGCCACGGACCTTCTCTGCA 102753 102772 -27.2 1090 305 CACGACAGTGCGGAAGAACA 102942 102961 -23.4 1091 306 TTCAGGGTGGTCCATGTCAC 103191 103210 -30.6 1092 307 GGTGCCTCACTAGCCCTAGC 103607 103626 -27.6 1093 308 TCGGTCCTCACCGTGACGGC 104008 104027 -23.7 1094 309 TCCCACTTCACATAAATATA 104510 104529 -26.9 1095 310 CATCAGACATCGTTTTGGAC 104872 104891 -25.8 1096 311 TAACACCCACGCATGGCTGC 105548 105567 -26.8 1097 312 CATAGACCAGAGCGCCAGTT 105710 105729 -30.3 1098 313 CTACCATAGACCAGAGCGCC 105714 105733 -33.2 1099 314 CCTGTGCCTATAATCCTGTC 106475 106494 -23.7 1100 315 CTGAAACCGTATTACTTTAC 106811 106830 -23.2 1101 316 ACTGAGATGAGGTCTTGCTC 107638 107657 -27.8 1102 317 GACGGGGTTTCACTGTGTTC 107892 107911 -22.4 1103 318 GCTAGAGGCCTGAGGCACTG 108781 108800 -31.9 1104 319 CTGCACAAGGCGGTGGAAGT 109251 109270 -26.1 1105 320 GCCCAAATTCAATCCGTGGC 110226 110245 -27.3 1106 321 TCTGATCTTCTGTGTATCTG 110268 110287 -27.6 1107 322 GCTCCAGACTTTAGGTAAGG 111305 111324 -27.6 1108 323 TCTTGGGTAGGGATTCGCAA 111502 111521 -27.7 1109 324 GCTTGTATGCGCTTGCCCTC 111709 111728 -32.2 1110 325 CCTACTTGGGAGTCAGTGTG 112167 112186 -30.9 1111 326 CCGCCTTGTGCGCACGTTCT 112372 112391 -31.5 1112 327 GGGTCATCAGCGAAAGGGGC 112485 112504 -24.9 1113 328 TCTTTTGTGAAGTGTCTGTT 112790 112809 -25.6 1114 329 CAGACCATACAAGGTCGTCC 113802 113821 -20.9 1115 330 TTGGCTTCATTTGAACTCCC 114144 114163 -16.6 1116 331 ATCAGGCAGGGAGCTTGCAG 114538 114557 -25.5 1117 332 GGCACTCCCCACATCTCAGA 115044 115063 -26.5 1118 333 CACCTCCCAGACAGGGTTGC 115075 115094 -28.5 1119 334 CCCAGACGGGGTGGCGGCCA 115474 115493 -31.3 1120 335 AGAGAGCACCCGGTTGGGGG 115697 115716 -33.9 1121 336 TTTATTTATTTATTTTATTT 116076 116095 -13.6 1122 337 GTTTCCGACTTTTAGCCCAA 116878 116897 -22.5 1123 338 CCACATGCACTGCACTGCTC 117480 117499 -29.6 1124 339 GGTACTTCCTCGCATACGCT 117817 117836 -27.5 1125 340 CAGAGGTAGGGTTTTGCCAT 117958 117977 -21.3 1126 341 TCCTTCACCAACTCTAAAAT 118437 118456 -30.9 1127 342 GTGCTCAAATGTTCGTCCTG 118606 118625 -22.3 1128 343 TAAATGCCTAGGACATTCCA 119013 119032 -25.5 1129 344 AAGGTGGACAGCCAGTCTCC 119318 119337 -27.8 1130 345 AACGGCAGCCAGGGTATCAA 119500 119519 -30.2 1131 346 GTCCAACGGCAGCCAGGGTA 119504 119523 -33.1 1132 347 GACTTGGAGTTTGGGCATGG 120118 120137 -31.7 1133 348 AGAAGTTAGTAAATAGTGAA 120332 120351 -26.3 1134 349 AACAAACTAGAGGGAAGTCT 121121 121140 -23.8 1135 350 AGAACCAGCAGAACCTTTTC 121291 121310 -23.9 1136 351 GTCTCCTGGGTAGCTGGAAT 121769 121788 -29 1137 352 GATTCTGCAAGCTTAGGTTA 122341 122360 -30.6 1138 353 AGATTCTGCAAGCTTAGGTT 122342 122361 -30.1 1139 354 AATTCTAGCTTTTGCAAAGG 122599 122618 -19.5 1140 355 ATTTTTCCCATTGAAAGGAA 123214 123233 -20.4 1141 356 TGAAAACTTCGTGCGGACCA 123381 123400 -28.5 1142 357 CCCAGACAGCAATTAGTACA 123796 123815 -23.5 1143 358 GTGAGACTGCAATCGGGTCC 123940 123959 -22 1144 359 TTTCATGTTGTCCGTCACTT 124538 124557 -23.7 1145 360 CTATCCTGTTGCGAGTTTTA 124788 124807 -16.2 1146 361 GTTTCACGGAAGAGCTATTA 124818 124837 -11.4 1147 362 CTTTTCTAAGAGTGTATAGG 125447 125466 -17.9 1148 363 GCTCTACCCAGCTATATGCC 125658 125677 -22.4 1149 364 GGGTAAACAGGTTTGCATCA 125818 125837 -25.4 1150 365 CCAGCTACGCTCAGTGGTCT 126034 126053 -21.9 1151 366 TTTTAAAGTATTTTATAATA 126231 126250 -15.4 1152 367 TGCAAGAGTACGCTTCAGCT 126564 126583 -29.7 1153 368 TCATACTACTGCACTATAAC 127217 127236 -24.4 1154 369 TACATTGTGTCAGGTGGATG 127795 127814 -27.3 1155 370 CTTCTAGACCATTAGGCCTG 127950 127969 -28.3 1156 371 CTCCTGTAGTCTATCTTCAA 128764 128783 -25.3 1157 372 ACTCACCCCAGTACGACACC 128853 128872 -28.6 1158 373 AGAACTCACCCCAGTACGAC 128856 128875 -29.6 1159 374 GGCACGTATCTGATGGACTC 128988 129007 -30.8 1160 375 AACAAAGCACGACCAGCCTA 129083 129102 -27.5 1161 376 TACTAGAAACGGGCAGTGTG 129149 129168 -24.5 1162 377 TTCTTCTCCCAAGTGAGGGG 129535 129554 -22.2 1163 378 GCCAAGAGCCGAACAACACA 129658 129677 -28.2 1164 379 AACTTAAAAGTATAATAATA 129855 129874 -17.4 1165 380 ACCTGCACGTTTTGCACATG 129883 129902 -26.4 1166 381 ACACTGTTGATGGGAGTCTA 130446 130465 -29.3 1167 382 GACACTTCTCAAAAGAAGAC 130636 130655 -24.3 1168 383 CAGACACTTCTCAAAAGAAG 130638 130657 -23 1169 384 TACAAAGAACTTAAACAAAT 130705 130724 -23.3 1170 385 ATCAGAGTGAGCAGGCAACT 130786 130805 -30.2 1171 386 GGCATAGGCATGGGCAAAGA 130906 130925 -28 1172 387 GCAGTGGGGAAAGGATTCCC 131092 131111 -25.2 1173 388 GAAAATGGCCATACTCCCCA 131425 131444 -27.8 1174 389 TTTGGAAAACCCCAACATTT 131739 131758 -20 1175 390 GCACAAGACAAGGATGCTCT 131901 131920 -28.3 1176 391 ACACTGGCAAACTGAATCCA 132237 132256 -26.2 1177 392 AAATGTCCAGGACCAGATGG 132470 132489 -24.5 1178 393 CTCTACTCAAATAAACTAGA 132623 132642 -20.8 1179 394 AGATCACCACTGATCCCACA 132679 132698 -22.2 1180 395 CTAGAGAAGCAAGAGCAAAC 132901 132920 -29.9 1181 396 GAACTAGAGAAGCAAGAGCA 132904 132923 -30.7 1182 397 AACAATGACACAGTGTACCC 133036 133055 -27.8 1183 398 ACTCTCCACCCCAAATCAAC 133318 133337 -27.7 1184 399 TGTGCTGTATTCAGGAGACA 133744 133763 -27.5 1185 400 GGAAAAAGCGGTACCAGCCA 133964 133983 -29.7 1186 401 ATGGTCAGATTCACGGAGGT 134439 134458 -26.4 1187 402 TGGATCAATCAAGCAGAAGA 134760 134779 -28.3 1188 403 GGTGAATTGCTAACTAGAAT 134868 134887 -18 1189 404 GAGCTCTGGCTGGAATCTGG 135382 135401 -29.8 1190 405 AGGGGCGGCTGTAGGCACAG 135575 135594 -24.6 1191 406 CGACCTGGGACGTTTGAGGT 135843 135862 -28.7 1192 407 GGGGTGTTGCCTTACTCGGG 136255 136274 -26.8 1193 408 GCTTATGATTACACGTGTTG 136514 136533 -25.8 1194 409 GCCAGGTGTCATGAAAAAAA 136812 136831 -27.2 1195 410 GAGGCAGGTCGTTGGATTCA 137470 137489 -23.7 1196 411 AAAGTGTTCACGACAATCAC 137742 137761 -26 1197 412 AAACATACGGTTTCAGACTG 138304 138323 -22.9 1198 413 GATAAAACATACGGTTTCAG 138308 138327 -24.7 1199 414 ACAAAACTTAATGCACTCAT 139058 139077 -21.2 1200 415 AAGAGTGAGTTTTGGAACAT 139584 139603 -25.5 1201 416 GGTGTACGGCGTGTGAGCGG 139844 139863 -35 1202 417 GGGTGGTGTACGGCGTGTGA 139848 139867 -34.2 1203 418 AGTTGAAGAAACCCTAAAGG 140000 140019 -23.1 1204 419 GTGTAAACGGCAGTTCCATG 140365 140384 -29.3 1205 420 ATGTGTAAACGGCAGTTCCA 140367 140386 -27.2 1206 421 CGGCTGCCGGTCATATGACT 2027 2046 -14.5 1207 422 CCGGCTGCCGGTCATATGAC 2028 2047 -17.3 1208 423 CTGCTCGGCGCCGGAGCCGC 2047 2066 -15.6 1209 424 GAGTAACTCTGCTCGGCGCC 2055 2074 -17.4 1210 425 CGCTGCGACTGAGTAACTCT 2065 2084 -29.4 1211 426 GCCGCTGCGACTGAGTAACT 2067 2086 -25.8 1212 427 GGCCGCTGCGACTGAGTAAC 2068 2087 -27.4 1213 428 GGCCGCCCAGCCTTGCGGGA 2094 2113 -19 1214 429 CGGCCGCCCAGCCTTGCGGG 2095 2114 -17.3 1215 430 CGCGGCCGCCCAGCCTTGCG 2097 2116 -14.8 1216 431 CGCGGTGGCGGGGCCATCGC 2122 2141 -14.1 1217 432 CCGCGGTGGCGGGGCCATCG 2123 2142 -20.5 1218 433 GGGGCCGGCGGCTCAGCGCG 2159 2178 -25.3 1219 434 TGGCGCTCGGGGGGCCGGCG 2169 2188 -19.7 1220 435 GGGCCATGATGCCGCCGCCG 2201 2220 -24 1221 436 GGGGCCATGATGCCGCCGCC 2202 2221 -22.6 1222 437 CGGGGCCATGATGCCGCCGC 2203 2222 -22.5 1223 438 TCGGGGCCATGATGCCGCCG 2204 2223 -24.1 1224 439 AGACGCCGCCGCGGAGCTGC 2263 2282 -13.7 1225 440 ACCTAAAGGCGGGTCCTTCC 2303 2322 -10.4 1226 441 CACCTAAAGGCGGGTCCTTC 2304 2323 -10 1227 442 TCACCTAAAGGCGGGTCCTT 2305 2324 -11.3 1228 443 CTCACCTAAAGGCGGGTCCT 2306 2325 -12.3 1229 444 CCTCACCTAAAGGCGGGTCC 2307 2326 -14.4 1230 445 CCCTCACCTAAAGGCGGGTC 2308 2327 -14.4 1231 446 CCCCTCACCTAAAGGCGGGT 2309 2328 -14.3 1232 447 TCAGGCGAATCTCATTCAGG 58938 58957 -22.2 1233 448 ATCAGGCGAATCTCATTCAG 58939 58958 -23.7 1234 449 TATCAGGCGAATCTCATTCA 58940 58959 -25.1 1235 450 CTATCAGGCGAATCTCATTC 58941 58960 -26 1236 451 ACTATCAGGCGAATCTCATT 58942 58961 -24.2 1237 452 AACTATCAGGCGAATCTCAT 58943 58962 -24.3 1238 453 AAACTATCAGGCGAATCTCA 58944 58963 -24.7 1239 454 TAAACTATCAGGCGAATCTC 58945 58964 -25.1 1240 455 GTAAACTATCAGGCGAATCT 58946 58965 -25.1 1241 456 TCAATCTTTTGAACAGCTTT 59008 59027 -15.4 1242 457 TCCCCCAGAAGAACTGTGGG 62168 62187 -21.1 1243 458 GACATCGGAAGCTGGTCTTG 64866 64885 -25.4 1244 459 TGACATCGGAAGCTGGTCTT 64867 64886 -23.6 1245 460 TTGACATCGGAAGCTGGTCT 64868 64887 -23.9 1246 461 TGTTGACATCGGAAGCTGGT 64870 64889 -25.1 1247 462 ATGTTGACATCGGAAGCTGG 64871 64890 -25.8 1248 463 CATGTTGACATCGGAAGCTG 64872 64891 -22.2 1249 464 ACATGTTGACATCGGAAGCT 64873 64892 -22.2 1250 465 TAAGGTGGAGCCTTTGTAAC 64926 64945 -23.8 1251 466 AGACTCACCGTATGTAGTGT 64951 64970 -24.3 1252 467 CCAGACTCACCGTATGTAGT 64953 64972 -25.6 1253 468 TCCTACACTTACTTATTTGT 65132 65151 -20.2 1254 469 CAAGCTATATCCGGAGATGG 66513 66532 -23.2 1255 470 GCAAGCTATATCCGGAGATG 66514 66533 -25.5 1256 471 TTGCAAGCTATATCCGGAGA 66516 66535 -26.7 1257 472 CTTGCAAGCTATATCCGGAG 66517 66536 -27.6 1258 473 TCTTGCAAGCTATATCCGGA 66518 66537 -26.7 1259 474 CAGTACGACAAGGACCCAAA 66598 66617 -27.5 1260 475 TACCTGATCGAGCAATGCCA 68461 68480 -25.7 1261 476 AGTACCTGATCGAGCAATGC 68463 68482 -25.1 1262 477 AAGTACCTGATCGAGCAATG 68464 68483 -25.1 1263 478 ACAAGTACCTGATCGAGCAA 68466 68485 -25.8 1264 479 CAAGCTGAATGTCTCATCAG 92063 92082 -18.6 1265 480 CCAAGCTGAATGTCTCATCA 92064 92083 -18.7 1266 481 GCCAAGCTGAATGTCTCATC 92065 92084 -18.2 1267 482 AGTGCCGGACATCATAGTAA 94396 94415 -24.4 1268 483 AAGTGCCGGACATCATAGTA 94397 94416 -24.1 1269 484 AAAGTGCCGGACATCATAGT 94398 94417 -24.9 1270 485 AGAGCTGGTTTTTTTCCAAA 94415 94434 -17.9 1271 486 CAGAGCTGGTTTTTTTCCAA 94416 94435 -17.9 1272 487 AACACCGCAGTCAGTAAGGC 94618 94637 -23.3 1273 488 TAACACCGCAGTCAGTAAGG 94619 94638 -23.1 1274 489 TTAACACCGCAGTCAGTAAG 94620 94639 -20 1275 490 GTTAACACCGCAGTCAGTAA 94621 94640 -20.2 1276 491 GGTTAACACCGCAGTCAGTA 94622 94641 -21 1277 492 AGGTTAACACCGCAGTCAGT 94623 94642 -20.9 1278 493 TAGGTTAACACCGCAGTCAG 94624 94643 -21.2 1279 494 GTAGGTTAACACCGCAGTCA 94625 94644 -21.9 1280 495 GGAGGTACGTTTCTCTGAGA 94700 94719 -19.6 1281 496 ATACGGATGTGTTTTGGCCA 94736 94755 -22.8 1282 497 TATACGGATGTGTTTTGGCC 94737 94756 -21.1 1283 498 TTATACGGATGTGTTTTGGC 94738 94757 -18.4 1284 499 ATTATACGGATGTGTTTTGG 94739 94758 -14.6 1285 500 AGAGGATTATACGGATGTGT 94744 94763 -16.3 1286 501 AAGAGGATTATACGGATGTG 94745 94764 -15.9 1287 502 AAAGAGGATTATACGGATGT 94746 94765 -17.4 1288 503 GCTCCGTAAAGATCACCTAG 101099 101118 -19.2 1289 504 GGCTCCGTAAAGATCACCTA 101100 101119 -21.4 1290 505 TGGCTCCGTAAAGATCACCT 101101 101120 -21.5 1291 506 ATGGCTCCGTAAAGATCACC 101102 101121 -25.7 1292 507 TATGGCTCCGTAAAGATCAC 101103 101122 -26.5 1293 508 CTATGGCTCCGTAAAGATCA 101104 101123 -26.2 1294 509 CCTATGGCTCCGTAAAGATC 101105 101124 -26.2 1295 510 CGGTGCGAGTCAGTCTCACT 101131 101150 -16.2 1296 511 ACGGTGCGAGTCAGTCTCAC 101132 101151 -20 1297 512 TACGGTGCGAGTCAGTCTCA 101133 101152 -20.8 1298 513 CTACGGTGCGAGTCAGTCTC 101134 101153 -22.4 1299 514 ACTACGGTGCGAGTCAGTCT 101135 101154 -21.9 1300 515 CCACTACGGTGCGAGTCAGT 101137 101156 -23.5 1301 516 ACCACTACGGTGCGAGTCAG 101138 101157 -22.3 1302 517 TACCACTACGGTGCGAGTCA 101139 101158 -14.1 1303 518 CTACCACTACGGTGCGAGTC 101140 101159 -16.1 1304 519 CCTACCACTACGGTGCGAGT 101141 101160 -17.7 1305 520 CCCTACCACTACGGTGCGAG 101142 101161 -16.2 1306 521 TCCCTACCACTACGGTGCGA 101143 101162 -15.5 1307 522 TTCCCTACCACTACGGTGCG 101144 101163 -15.8 1308 523 AGTATTCGCTGGACTAAGTC 101171 101190 -24 1309 524 AAGTATTCGCTGGACTAAGT 101172 101191 -21.6 1310 525 AAAGTATTCGCTGGACTAAG 101173 101192 -22.1 1311 526 TAAAGTATTCGCTGGACTAA 101174 101193 -19.6 1312 527 ATAAAGTATTCGCTGGACTA 101175 101194 -20 1313 528 CATAAAGTATTCGCTGGACT 101176 101195 -19.4 1314 529 GACATAAAGTATTCGCTGGA 101178 101197 -19.4 1315 530 GGACATAAAGTATTCGCTGG 101179 101198 -21.1 1316 531 GTAGCTCAGAGCAACGGAGA 101209 101228 -14.8 1317 532 TTCCTCACCGTAATGACCAC 101339 101358 -25.6 1318 533 GTTCCTCACCGTAATGACCA 101340 101359 -24.9 1319 534 CGTTCCTCACCGTAATGACC 101341 101360 -26.2 1320 535 TCGTTCCTCACCGTAATGAC 101342 101361 -24.5 1321 536 CTCGTTCCTCACCGTAATGA 101343 101362 -23.4 1322 537 GCTCGTTCCTCACCGTAATG 101344 101363 -24.7 1323 538 GGCTCGTTCCTCACCGTAAT 101345 101364 -25.5 1324 539 GGTACAAGAGCGGGCTCGTT 101357 101376 -25.4 1325 540 GGGTACAAGAGCGGGCTCGT 101358 101377 -26.8 1326 541 GGGGTACAAGAGCGGGCTCG 101359 101378 -29 1327 542 GAAGCGTCTGATGCTGGCCC 101540 101559 -18.5 1328 543 GGAAGCGTCTGATGCTGGCC 101541 101560 -20.5 1329 544 AGGAAGCGTCTGATGCTGGC 101542 101561 -20.7 1330 545 CAGGAAGCGTCTGATGCTGG 101543 101562 -19.2 1331 546 CCAGGAAGCGTCTGATGCTG 101544 101563 -17.3 1332 547 TCCAGGAAGCGTCTGATGCT 101545 101564 -21 1333 548 TTCCAGGAAGCGTCTGATGC 101546 101565 -22.3 1334 549 GGTTTCCAGGAAGCGTCTGA 101549 101568 -23.6 1335 550 AGAGCCATCTTGCGGTGCCT 101604 101623 -26.5 1336 551 AAGAGCCATCTTGCGGTGCC 101605 101624 -26.9 1337 552 CACTGACCGGTCTGGCAGTC 101715 101734 -25.8 1338 553 CCACTGACCGGTCTGGCAGT 101716 101735 -23.1 1339 554 GGCCACTGACCGGTCTGGCA 101718 101737 -23.7 1340 555 AAGCTTCCAATCTGGAAAGT 101789 101808 -18 1341 556 ATTTTTTTCATGCGGCTTTC 101831 101850 -20.1 1342 557 CATTTTTTTCATGCGGCTTT 101832 101851 -22.2 1343 558 CCATTTTTTTCATGCGGCTT 101833 101852 -24.4 1344 559 TCCATTTTTTTCATGCGGCT 101834 101853 -25.1 1345 560 CTCCATTTTTTTCATGCGGC 101835 101854 -24.9 1346 561 GGCCTTCACCCGTTCCTCCA 101850 101869 -25.4 1347 562 AGGCCTTCACCCGTTCCTCC 101851 101870 -24.7 1348 563 AGGGGCCACAGGCCTTCACC 101860 101879 -12.2 1349 564 GAGGGGCCACAGGCCTTCAC 101861 101880 -12.8 1350 565 GGAGGGGCCACAGGCCTTCA 101862 101881 -12.7 1351 566 ACCCTTGTCAGACTCGCCTT 102000 102019 -26.4 1352 567 AACCCTTGTCAGACTCGCCT 102001 102020 -26.6 1353 568 ACCTGGGGGCAACAGGCTCC 102187 102206 -19.2 1354 569 CACCTGGGGGCAACAGGCTC 102188 102207 -20 1355 570 GGGGATATTCGCACCAGCCA 102231 102250 -21.2 1356 571 AGGGGATATTCGCACCAGCC 102232 102251 -21.8 1357 572 CAGGGGATATTCGCACCAGC 102233 102252 -23 1358 573 ACAGGGGATATTCGCACCAG 102234 102253 -23 1359 574 CACAGGGGATATTCGCACCA 102235 102254 -23.8 1360 575 CTCCCAGGGCGCTATCTGAG 102313 102332 -27.6 1361 576 TCTCCCAGGGCGCTATCTGA 102314 102333 -26.8 1362 577 CAGGCTTCGTCGTCACTGTC 102332 102351 -25.1 1363 578 GCAGGCTTCGTCGTCACTGT 102333 102352 -24.9 1364 579 CGCAGGCTTCGTCGTCACTG 102334 102353 -25.5 1365 580 AAGCGCAGGCTTCGTCGTCA 102337 102356 -27.5 1366 581 GCTCCACTTCCAAGGACCCT 102397 102416 -20 1367 582 AGCTCCACTTCCAAGGACCC 102398 102417 -17.2 1368 583 GCCCGCCAGAAGTGAGCGGC 103352 103371 -22.1 1369 584 AGCCCGCCAGAAGTGAGCGG 103353 103372 -16.3 1370 585 GTCTGCGATAATACAGACAG 124478 124497 -16.3 1371 586 TGTCTGCGATAATACAGACA 124479 124498 -14.4 1372 587 TTATCCGTGTCTGCGATAAT 124486 124505 -17.9 1373 588 TTTATCCGTGTCTGCGATAA 124487 124506 -19.1 1374 589 ATTTATCCGTGTCTGCGATA 124488 124507 -19.8 1375 590 AGACCAGGACATCCTGGCCT 124560 124579 -11.8 1376 591 TGTAGTCTATCTTCAAGATG 128760 128779 -25.2 1377 592 AGTACGACACCTAATTCTTT 128844 128863 -22.1 1378 593 CAGTACGACACCTAATTCTT 128845 128864 -22.6 1379 594 CCAGTACGACACCTAATTCT 128846 128865 -23.1 1380 595 CCCAGTACGACACCTAATTC 128847 128866 -23.8 1381 596 CCCCAGTACGACACCTAATT 128848 128867 -24.5 1382 597 ACCCCAGTACGACACCTAAT 128849 128868 -26.2 1383 598 CACCCCAGTACGACACCTAA 128850 128869 -26.4 1384 599 TCACCCCAGTACGACACCTA 128851 128870 -27.2 1385 600 CTCACCCCAGTACGACACCT 128852 128871 -26 1386 601 AACTCACCCCAGTACGACAC 128854 128873 -26.3 1387 602 GAACTCACCCCAGTACGACA 128855 128874 -28.3 1388 603 GGTCGTTGGATTCAATCCTG 137464 137483 -20 1389 604 AGGTCGTTGGATTCAATCCT 137465 137484 -18.6 1390 605 CAGGTCGTTGGATTCAATCC 137466 137485 -20.9 1391 606 GCAGGTCGTTGGATTCAATC 137467 137486 -20.8 1392 607 GGCAGGTCGTTGGATTCAAT 137468 137487 -21.2 1393 608 AGGCAGGTCGTTGGATTCAA 137469 137488 -21.4 1394 609 AGAGGCAGGTCGTTGGATTC 137471 137490 -23.3 1395 610 CAGAGAGCTTATTCCTTTCT 137603 137622 -16.7 1396 611 ATTATGTCAAGTAAACCTAA 137715 137734 -20.7 1397 612 AGTGTTCACGACAATCACAA 137740 137759 -25.1 1398 613 AAGTGTTCACGACAATCACA 137741 137760 -25.5 1399 614 TAAAGTGTTCACGACAATCA 137743 137762 -24.5 1400 615 CTAAAGTGTTCACGACAATC 137744 137763 -25.5 1401 616 CCTAAAGTGTTCACGACAAT 137745 137764 -23.1 1402 617 AGCAGTTTCCTACTGGAATA 137918 137937 -13.1 1403 618 ATGTTTTCTCCAGTTTGGGG 137958 137977 -19.4 1404 619 TATGTTTTCTCCAGTTTGGG 137959 137978 -20 1405 620 TTTATTGCACCAACAATCCA 138224 138243 -11.3 1406 621 GTTTATTGCACCAACAATCC 138225 138244 -15.5 1407 622 GGTTTATTGCACCAACAATC 138226 138245 -17.4 1408 623 GGGTTTATTGCACCAACAAT 138227 138246 -14.3 1409 624 CATACGGTTTCAGACTGTAC 138301 138320 -21.4 1410 625 ACATACGGTTTCAGACTGTA 138302 138321 -20.8 1411 626 AACATACGGTTTCAGACTGT 138303 138322 -21.7 1412 627 ATAAAACATACGGTTTCAGA 138307 138326 -21.7 1413 628 CCAACCGAAACACTCTTGAG 138393 138412 -10.3 1414 629 ACCAACCGAAACACTCTTGA 138394 138413 -10.6 1415 630 TACCAACCGAAACACTCTTG 138395 138414 -11 1416 631 TTACCAACCGAAACACTCTT 138396 138415 -9.4 1417 632 AACTTACCAACCGAAACACT 138399 138418 -8.4 1418 633 GAACTTACCAACCGAAACAC 138400 138419 -11.1 1419 634 AGAACTTACCAACCGAAACA 138401 138420 -9.4 1420 635 AATGATAATATAAAAATTTA 138576 138595 -6.9 1421 636 AAATGATAATATAAAAATTT 138577 138596 -3.8 1422 637 GAAATGATAATATAAAAATT 138578 138597 -6.9 1423 638 AAAAGTCTTTTACTTTACCC 138785 138804 -19.2 1424 639 TTTAAAAGTCTTTTACTTTA 138788 138807 -11.8 1425 640 ATGACCAATATTTAAAAGTC 138798 138817 -17.6 1426 641 GCAAAGTGCTAAAGATGTTT 138847 138866 -18.9 1427 642 TGCAAAGTGCTAAAGATGTT 138848 138867 -18.9 1428 643 GAACTGGAATTAATTCATAA 138910 138929 -19.3 1429 644 AAAACTTAATGCACTCATCT 139056 139075 -19.6 1430 645 CAAAACTTAATGCACTCATC 139057 139076 -20.5 1431 646 AGAATGAACATTTCAATTTT 139108 139127 -16.2 1432 647 AAGAATGAACATTTCAATTT 139109 139128 -15.4 1433 648 TGTCCCCTATATGGGTCTAA 139469 139488 -20.4 1434 649 GTGTCCCCTATATGGGTCTA 139470 139489 -23 1435 650 AGTGTCCCCTATATGGGTCT 139471 139490 -22.6 1436 651 GGCATTTTTCCAAGAGTGAG 139595 139614 -24.3 1437 652 ATTTAAAGGATTATAAAAGA 139732 139751 -15.8 1438 653 TGTGAGCGGTGGATCTCAAC 139833 139852 -22.5 1439 654 GTGTGAGCGGTGGATCTCAA 139834 139853 -25.5 1440 655 CGTGTGAGCGGTGGATCTCA 139835 139854 -26.6 1441 656 GCGTGTGAGCGGTGGATCTC 139836 139855 -29.3 1442 657 GGCGTGTGAGCGGTGGATCT 139837 139856 -31.4 1443 658 ACGGCGTGTGAGCGGTGGAT 139839 139858 -34.5 1444 659 TACGGCGTGTGAGCGGTGGA 139840 139859 -33.1 1445 660 GTACGGCGTGTGAGCGGTGG 139841 139860 -35 1446 661 TGTACGGCGTGTGAGCGGTG 139842 139861 -32.9 1447 662 GTGTACGGCGTGTGAGCGGT 139843 139862 -34.2 1448 663 TGGTGTACGGCGTGTGAGCG 139845 139864 -34.3 1449 664 GTGGTGTACGGCGTGTGAGC 139846 139865 -33.6 1450 665 GGTGGTGTACGGCGTGTGAG 139847 139866 -31.7 1451 666 TGGGTGGTGTACGGCGTGTG 139849 139868 -34.2 1452 667 CTGGGTGGTGTACGGCGTGT 139850 139869 -31.9 1453 668 CACTGGGTGGTGTACGGCGT 139852 139871 -29.5 1454 669 CCTCTGCGGCTAAGCCAGAA 139878 139897 -20.1 1455 670 GCCTCTGCGGCTAAGCCAGA 139879 139898 -24 1456 671 TGCCTCTGCGGCTAAGCCAG 139880 139899 -22.6 1457 672 TTTAAAAAAAATTGTTTTAT 139944 139963 -6.8 1458 673 TTTTAAAAAAAATTGTTTTA 139945 139964 -6 1459 674 TGAAGAAACCCTAAAGGTGG 139997 140016 -22.5 1460 675 TTAAAAGTTGAAGAAACCCT 140005 140024 -18.5 1461 676 TTTACAGACTTCTGCTCTGT 140088 140107 -13.4 1462 677 ATTTACAGACTTCTGCTCTG 140089 140108 -14.7 1463 678 CCAATTTATTAAAAATAAAA 140144 140163 -10.3 1464 679 ACCAATTTATTAAAAATAAA 140145 140164 -11.6 1465 680 TACACAATTACCTTTACATT 140175 140194 -18.5 1466 681 CGTACACAATTACCTTTACA 140177 140196 -19.6 1467 682 CATTTTAAATTTTCATTGTC 140207 140226 -8.3 1468 683 GATAAAAAATAAAATACTTT 140253 140272 -10.3 1469 684 TGATAAAAAATAAAATACTT 140254 140273 -12 1470 685 TTGATAAAAAATAAAATACT 140255 140274 -11.8 1471 686 ATTGATAAAAAATAAAATAC 140256 140275 -11.1 1472 687 ACTGATTGATAAAAAATAAA 140260 140279 -12.6 1473 688 AAACAGCACACTTCGAAAAA 140312 140331 -17.4 1474 689 TTACTTTCATTAAAAAACAG 140326 140345 -20.2 1475 690 TGTGTAAACGGCAGTTCCAT 140366 140385 -27.7 1476 691 ACAACGGTTTAGTATCATTT 140457 140476 -17.1 1477 692 AGAGCCCAAACAACGGTTTA 140466 140485 -16.8 1478 693 ATTAGCGGAAAAATCCAGAC 140603 140622 -22.9 1479 694 TATTAGCGGAAAAATCCAGA 140604 140623 -20.3 1480 695 AAGTACATATTAGCGGAAAA 140611 140630 -15.7 1481 696 AAAGTACATATTAGCGGAAA 140612 140631 -15.5 1482 697 TTCTCTAAAGTACATATTAG 140618 140637 -10.3 1483 698 ATTCTCTAAAGTACATATTA 140619 140638 -8.5 1484 699 AATATTCTCTAAAGTACATA 140622 140641 -8.6 1485 700 ATGAACAAAATATTCTCTAA 140630 140649 -14.8 1486 701 CATGAACAAAATATTCTCTA 140631 140650 -16.3 1487 702 AGTATGCATGAACAAAATAT 140637 140656 -13.9 1488 703 AATTATATTAAAATACCCTC 140732 140751 -16 1489 704 AAAATTATATTAAAATACCC 140734 140753 -13.5 1490 705 AAAAATTATATTAAAATACC 140735 140754 -12.4 1491 706 TCAAGATATATCGTTCATCA 140805 140824 -20.8 1492 707 TTCAAGATATATCGTTCATC 140806 140825 -20.5 1493 708 TTTCAAGATATATCGTTCAT 140807 140826 -19 1494

TABLE 5 Example antisense oligonucleotide modulators to downregulate FNIP2 Target start Target stop site with site with reference to reference to SEQ ID SEQ ID SEQ ID GI ID# Sequence NO: 1 NO: 1 NO GiTx-66 GTGGATAGCGGAAATGGTGG   3939   3958  804 GiTx-67 GGTGT ACGGCGTGTG AGCGG 139844 139863 1202 GiTx-68 CCTGTCTACTCCGGTGTGTT  39376  39395  912 GiTx-69 TTGTGTTGGGACCGTGACCC  23023  23042  865 GiTx-70 CTCTACTCTCACTGGCTTGT  64249  64268  975 GiTx-71 CCGCT GCGACTGAGT AACTC   2066   2085  787 GiTx-72 TCCTC ACCGTAATGA CCACA 101338 101357 1083 GiTx-73 GTTGA CATCGGAAGC TGGTC  64869  64888  978 GiTx-74 ACTGA CCGGTCTGGC AGTCT 101714 101733 1085

TABLE 6 Example antisense oligonucleotide modulators to upregulate FNIP2 Target start Target stop site with site with reference to reference to SEQ ID SEQ ID SEQ ID GI ID# Sequence NO: 1 NO: 1 NO GiTx-75 ATGTGTAAACGGCAGTTCCA 140367 140386 1206 GiTx-76 GTGTAAACGGCAGTTCCATG 140365 140384 1205 GiTx-77 ACAAAACTTAATGCACTCAT 139058 139077 1200 GiTx-78 GATAAAACATACGGTTTCAG 138308 138327 1199 GiTx-79 AAACATACGGTTTCAGACTG 138304 138323 1198 GiTx-80 AAAGTGTTCACGACAATCAC 137742 137761 1197

TABLE 7 Human iPSC Lines for Evaluating Therapies for Neurodegenerative Disease such as ALS Name Type ALS Subtype/Mutation GI-iPSC 1 Healthy NA GI-iPSC 2 Isogenic TARDBP (G298S) GI-iPSC 3 Isogenic SOD1 (L144F) GI-iPSC 4 Patient Sporadic GI-iPSC 5 Patient C9ORF72 GI-iPSC 6 Patient TARDBP GI-iPSC 7 Patient Sporadic GI-iPSC 8 Patient C9ORF72 GI-iPSC 9 Patient SOD1 (A4V) GI-iPSC 10 Healthy NA GI-iPSC 11 Healthy NA

TABLE 8 Normalized Survival Index (NSI) of Human ALS iPSC-derived Motor Neurons Day 28 Day 31 Cell line Treatment NSI STDEV NSI STDEV BJ-iPS si-NT 1.00 0.06 1.07 0.09 si-FLCN 1.00 0.07 BJ-SOD1 (L144F) si-NT 0.96 0.05 0.53 0.02 si-FLCN 0.89 0.02 BJ-TDP43 (G298S) si-NT 0.91 0.03 0.48 0.08 si-FLCN 0.73 0.05 CS14isALS-Tn16 si-NT 0.93 0.05 0.48 0.06 si-FLCN 0.96 0.10

TABLE 9 Phosphorylated TDP-43 (pTDP43) levels in Human ALS iPSC-derived Motor Neurons Day 28 Day 31 Cell line Treatment pTDP43 STDEV pTDP43 STDEV BJ-iPS si-NT 1.00 0.03 1.05 0.02 si-FLCN 0.97 0.01 BJ-SOD1 (L144F) si-NT 8.51 0.87 8.25 0.17 si-FLCN 4.68 0.15 BJ-TDP43 (G298S) si-NT 8.04 0.65 7.58 0.40 si-FLCN 4.70 0.54 CS14isALS-Tn16 si-NT 9.74 0.62 9.50 0.27 si-FLCN 5.58 0.43

TABLE 10 miRNA modulators targeting SEQ ID NO: 11 Example seed location with reference to  Target SEQ ID No Name miRNA Sequence Score NO: 11 1 hsa-miR-32-5p UAUUGCACAUUACUAAGUUGCA 100 156175 2 hsa-miR-92a-3p UAUUGCACUUGUCCCGGCCUGU 100 156175 3 hsa-miR-92b-3p UAUUGCACUCGUCCCGGCCUCC 100 156175 4 hsa-miR-641 AAAGACAUAGGAUAGAGUCACCUC 100 157002 5 hsa-miR-363-3p AAUUGCACGGUAUCCAUCUGUA 100 156175 6 hsa-miR-3617-5p AAAGACAUAGUUGCAAGAUGGG 100 157002 7 hsa-miR-4495 AAUGUAAACAGGCUUUUUGCU 99 155144 8 hsa-miR-25-3p CAUUGCACUUGUCUCGGUCUGA 99 156175 9 hsa-miR-367-3p AAUUGCACUUUAGCAAUGGUGA 99 156175 10 hsa-miR-12129 GAUGUACUGAACUGGGUCAGAC 98 154889 11 hsa-miR-186-5p CAAAGAAUUCUCCUUUUGGGCU 97 154403 12 hsa-miR-1272 GAUGAUGAUGGCAGCAAAUUCUGAAA 97 155643 13 hsa-miR-4773 CAGAACAGGAGCAUAGAAAGGC 96 155123 14 hsa-miR-6799-5p GGGGAGGUGUGCAGGGCUGG 96 156211 15 hsa-miR-190a-3p CUAUAUAUCAAACAUAUUCCU 96 154959 16 hsa-miR-5680 GAGAAAUGCUGGACUAAUCUGC 96 154598 17 hsa-miR-4325 UUGCACUUGUCUCAGUGA 95 156174 18 hsa-miR-300 UAUACAAGGGCAGACUCUCUCU 94 154465 19 hsa-miR-374b-5p AUAUAAUACAACCUGCUAAGUG 94 154835 20 hsa-miR-374a-5p UUAUAAUACAACCUGAUAAGUG 94 154835 21 hsa-miR-539-5p GGAGAAAUUAUCCUUGGUGUGU 94 154801 22 hsa-miR-4500 UGAGGUAGUAGUUUCUU 93 155535 23 hsa-miR-580-3p UUGAGAAUGAUGAAUCAUUAGG 93 154803 24 hsa-miR-1199-5p CCUGAGCCCGGGCCGCGCAG 93 154689 25 hsa-miR-369-3p AAUAAUACAUGGUUGAUCUUU 93 157235 26 hsa-miR-7-2-3p CAACAAAUCCCAGUCUACCUAA 93 154761 27 hsa-miR-7-1-3p CAACAAAUCACAGUCUGCCAUA 93 154761 28 hsa-miR-6751-3p ACUGAGCCUCUCUCUCUCCAG 93 154689 29 hsa-miR-4303 UUCUGAGCUGAGGACAG 92 154690 30 hsa-miR-218-1-3p AUGGUUCCGUCAAGCACCAUGG 92 155470 31 hsa-miR-381-3p UAUACAAGGGCAAGCUCUCUGU 92 154465 32 hsa-miR-6832-3p ACCCUUUUUCUCUUUCCCAG 92 156909 33 hsa-let-7c-5p UGAGGUAGUAGGUUGUAUGGUU 91 155535 34 hsa-let-7e-5p UGAGGUAGGAGGUUGUAUAGUU 91 155535 35 hsa-let-7b-5p UGAGGUAGUAGGUUGUGUGGUU 91 155535 36 hsa-miR-510-5p UACUCAGGAGAGUGGCAAUCAC 91 155936 37 hsa-let-7f-5p UGAGGUAGUAGAUUGUAUAGUU 91 155535 38 hsa-let-7i-5p UGAGGUAGUAGUUUGUGCUGUU 91 155535 39 hsa-let-7g-5p UGAGGUAGUAGUUUGUACAGUU 91 155535 40 hsa-let-7a-5p UGAGGUAGUAGGUUGUAUAGUU 91 155535 41 hsa-miR-98-5p UGAGGUAGUAAGUUGUAUUGUU 91 155535 42 hsa-miR-4458 AGAGGUAGGUGUGGAAGAA 90 155535 43 hsa-miR-6719-3p UCUGACAUCAGUGAUUCUCCUG 90 154480 44 hsa-let-7d-5p AGAGGUAGUAGGUUGCAUAGUU 90 155535 45 hsa-miR-3133 UAAAGAACUCUUAAAACCCAAU 90 155223 46 hsa-miR-95-5p UCAAUAAAUGUCUGUUGAAUU 90 156440 47 hsa-miR-6715a-3p CCAAACCAGUCGUGCCUGUGG 90 156967 48 hsa-miR-590-3p UAAUUUUAUGUAUAAGCUAGU 90 155173 49 hsa-miR-4728-5p UGGGAGGGGAGAGGCAGCAAGCA 89 155372 50 hsa-miR-323a-3p CACAUUACACGGUCGACCUCU 89 155488 51 hsa-miR-5580-3p CACAUAUGAAGUGAGCCAGCAC 89 154892 52 hsa-miR-4310 GCAGCAUUCAUGUCCC 89 154989 53 hsa-miR-579-3p UUCAUUUGGUAUAAACCGCGAUU 88 154536 54 hsa-miR-1-5p ACAUACUUCUUUAUAUGCCCAU 88 155114 55 hsa-miR-411-3p UAUGUAACACGGUCCACUAACC 88 155145 56 hsa-miR-589-3p UCAGAACAAAUGCCGGUUCCCAGA 88 157151 57 hsa-miR-7157-5p UCAGCAUUCAUUGGCACCAGAGA 88 154989 58 hsa-miR-664b-3p UUCAUUUGCCUCCCAGCCUACA 88 154536 59 hsa-miR-4775 UUAAUUUUUUGUUUCGGUCACU 88 154542 60 hsa-miR-7162-3p UCUGAGGUGGAACAGCAGC 88 155537 61 hsa-miR-7152-5p UUUCCUGUCCUCCAACCAGACC 88 156898 62 hsa-miR-7106-5p UGGGAGGAGGGGAUCUUGGG 88 156211 63 hsa-miR-379-3p UAUGUAACAUGGUCCACUAACU 88 155145 64 hsa-miR-499a-5p UUAAGACUUGCAGUGAUGUUU 88 157004 65 hsa-miR-3664-3p UCUCAGGAGUAAAGACAGAGUU 88 156647 66 hsa-miR-188-5p CAUCCCUUGCAUGGUGGAGGG 87 156911 67 hsa-miR-6729-3p UCAUCCCCCUCGCCCUCUCAG 87 157086 68 hsa-miR-196a-5p UAGGUAGUUUCAUGUUGUUGGG 87 155534 69 hsa-miR-196b-5p UAGGUAGUUUCCUGUUGUUGGG 87 155534 70 hsa-miR-137-3p UUAUUGCUUAAGAAUACGCGUAG 87 157034 71 hsa-miR-6785-5p UGGGAGGGCGUGGAUGAUGGUG 87 155372 72 hsa-miR-5011-5p UAUAUAUACAGCCAUGCACUC 87 154959 73 hsa-miR-4482-3p UUUCUAUUUCUCAGUGGGGCUC 87 154923 74 hsa-miR-493-5p UUGUACAUGGUAGGCUUUCAUU 87 157186 75 hsa-miR-3613-3p ACAAAAAAAAAAGCCCAACCCUUC 86 154498 76 hsa-miR-6758-3p ACUCAUUCUCCUCUGUCCAG 86 154431 77 hsa-miR-374c-5p AUAAUACAACCUGCUAAGUGCU 85 157234 78 hsa-miR-208a-3p AUAAGACGAGCAAAAAGCUUGU 85 157004 79 hsa-miR-655-3p AUAAUACAUGGUUAACCUCUUU 85 157234 80 hsa-miR-208b-3p AUAAGACGAACAAAAGGUUUGU 85 157004 81 hsa-miR-3658 UUUAAGAAAACACCAUGGAGAU 85 155341 82 hsa-miR-6883-5p AGGGAGGGUGUGGUAUGGAUGU 84 155372 83 hsa-miR-4666a-3p CAUACAAUCUGACAUGUAUUU 84 154464 84 hsa-miR-3189-3p CCCUUGGGUCUGAUGGGGUAG 84 154370 85 hsa-miR-548o-3p CCAAAACUGCAGUUACUUUUGC 84 154768 86 hsa-miR-149-3p AGGGAGGGACGGGGGCUGUGC 84 155372 87 hsa-miR-1323 UCAAAACUGAGGGGCAUUUUCU 84 154768 88 hsa-miR-6866-3p GAUCCCUUUAUCUGUCCUCUAG 83 156911 89 hsa-miR-3177-5p UGUGUACACACGUGCCAGGCGCU 82 157226 90 hsa-miR-548aw GUGCAAAAGUCAUCACGGUU 82 154770 91 hsa-miR-659-3p CUUGGUUCAGGGAGGGUCCCCA 82 155471 92 hsa-miR-4280 GAGUGUAGUUCUGAGCAGAGC 82 155326 93 hsa-miR-6780a-5p UUGGGAGGGAAGACAGCUGGAGA 81 156211 94 hsa-miR-3119 UGGCUUUUAACUUUGAUGGC 81 154575 95 hsa-miR-1245b-3p UCAGAUGAUCUAAAGGCCUAUA 80 154426 96 hsa-miR-7703 UUGCACUCUGGCCUUCUCCCAGG 80 156173 97 hsa-miR-5692b AAUAAUAUCACAGUAGGUGU 80 154613 98 hsa-miR-5692c AAUAAUAUCACAGUAGGUGUAC 80 154613 99 hsa-miR-211-5p UUCCCUUUGUCAUCCUUCGCCU 80 156910 100 hsa-miR-204-5p UUCCCUUUGUCAUCCUAUGCCU 80 156910 101 hsa-miR-4682 UCUGAGUUCCUGGAGCCUGGUCU 79 154844 102 hsa-miR-219b-3p AGAAUUGCGUUUGGACAAUCAGU 78 154774 103 hsa-miR-380-3p UAUGUAAUAUGGUCCACAUCUU 78 155145 104 hsa-miR-944 AAAUUAUUGUACAUCGGAUGAG 78 154555 105 hsa-miR-3120-3p CACAGCAAGUGUAGACAGGCA 78 155729 106 hsa-miR-6507-5p GAAGAAUAGGAGGGACUUUGU 77 155221 107 hsa-miR-541-5p AAAGGAUUCUGCUGUCGGUCCCACU 77 156633 108 hsa-miR-1260b AUCCCACCACUGCCACCAU 77 154350 109 hsa-miR-1260a AUCCCACCUCUGCCACCA 77 154350 110 hsa-miR-98-3p CUAUACAACUUACUACUUUCCC 76 154465 111 hsa-let-7b-3p CUAUACAACCUACUGCCUUCCC 76 154465 112 hsa-let-7f-1-3p CUAUACAAUCUAUUGCCUUCCC 76 154465 113 hsa-let-7a-3p CUAUACAAUCUACUGUCUUUC 76 154465 114 hsa-miR-30c-1-3p CUGGGAGAGGGUUGUUUACUCC 75 155942 115 hsa-miR-6788-5p CUGGGAGAAGAGUGGUGAAGA 75 155942 116 hsa-miR-607 GUUCAAAUCCAGAUCUAUAAC 75 154569 117 hsa-miR-30b-3p CUGGGAGGUGGAUGUUUACUUC 75 156211 118 hsa-miR-664a-3p UAUUCAUUUAUCCCCAGCCUACA 75 154608 119 hsa-miR-6779-5p CUGGGAGGGGCUGGGUUUGGC 75 156211 120 hsa-miR-3689a-3p CUGGGAGGUGUGAUAUCGUGGU 75 156211 121 hsa-miR-1273h-5p CUGGGAGGUCAAGGCUGCAGU 75 156211 122 hsa-miR-3689b-3p CUGGGAGGUGUGAUAUUGUGGU 75 156211 123 hsa-miR-548aj-3p UAAAAACUGCAAUUACUUUUA 75 154711 124 hsa-miR-3689c CUGGGAGGUGUGAUAUUGUGGU 75 156211 125 hsa-miR-30c-2-3p CUGGGAGAAGGCUGUUUACUCU 75 155942 126 hsa-miR-340-5p UUAUAAAGCAAUGAGACUGAUU 75 155095 127 hsa-miR-6736-3p UCAGCUCCUCUCUACCCACAG 75 155286 128 hsa-miR-548x-3p UAAAAACUGCAAUUACUUUC 75 154711 129 hsa-miR-494-3p UGAAACAUACACGGGAAACCUC 74 155021 130 hsa-miR-12119 UUCUGAGGGGACGGUAGAUUUGGGG 74 154691 131 hsa-miR-212-5p ACCUUGGCUCUAGACUGCUUACU 73 154370 132 hsa-miR-548c-3p CAAAAAUCUCAAUUACUUUUGC 73 154526 133 hsa-miR-553 AAAACGGUGAGAUUUUGUUUU 73 156524 134 hsa-miR-3674 AUUGUAGAACCUAAGAUUGGCC 73 154601 135 hsa-miR-548ah-3p CAAAAACUGCAGUUACUUUUGC 72 154711 136 hsa-miR-548am-3p CAAAAACUGCAGUUACUUUUGU 72 154711 137 hsa-miR-548aq-3p CAAAAACUGCAAUUACUUUUGC 72 154711 138 hsa-miR-1224-5p GUGAGGACUCGGGAGGUGG 72 155317 139 hsa-miR-548j-3p CAAAAACUGCAUUACUUUUGC 72 154711 140 hsa-miR-548ae-3p CAAAAACUGCAAUUACUUUCA 72 154711 141 hsa-miR-337-3p CUCCUAUAUGAUGCCUUUCUUC 72 156639 142 hsa-miR-3923 AACUAGUAAUGUUGGAUUAGGG 72 154974 143 hsa-miR-1245a AAGUGAUCUAAAGGCCUACAU 71 154634 144 hsa-miR-6807-5p GUGAGCCAGUGGAAUGGAGAGG 70 154688 145 hsa-miR-9898 UACUUACCUGUCCCCUACCCCA 70 154811 146 hsa-miR-3529-3p AACAACAAAAUCACUAGUCUUCCA 70 155851 147 hsa-miR-655-5p AGAGGUUAUCCGUGUUAUGUUC 70 157244 148 hsa-miR-23b-3p AUCACAUUGCCAGGGAUUACCAC 69 156807 149 hsa-miR-23a-3p AUCACAUUGCCAGGGAUUUCC 69 156807 150 hsa-miR-10523-5p GACAAUGAUGAGAAGACCUGAGGA 69 154463 151 hsa-miR-8079 CAGUGAUCGUCUCUGCUGGC 69 154634 152 hsa-miR-23c AUCACAUUGCCAGUGAUUACCC 69 156807 153 hsa-miR-2052 UGUUUUGAUAACAGUAAUGU 68 154779 154 hsa-miR-12123 UUAUUCAUUCACAAAAGCUUUA 68 154609 155 hsa-miR-4764-5p UGGAUGUGGAAGGAGUUAUCU 68 155718 156 hsa-miR-202-3p AGAGGUAUAGGGCAUGGGAA 67 155536 157 hsa-miR-3202 UGGAAGGGAGAAGAGCUUUAAU 67 155376 158 hsa-miR-223-5p CGUGUAUUUGACAAGCUGAGUU 66 157278 159 hsa-miR-4672 UUACACAGCUGGACAGAGGCA 66 154474 160 hsa-miR-502-5p AUCCUUGCUAUCUGGGUGCUA 66 154371 161 hsa-miR-5590-3p AAUAAAGUUCAUGUAUGGCAA 65 155094 162 hsa-miR-5584-5p CAGGGAAAUGGGAAGAACUAGA 65 154409 163 hsa-miR-142-5p CAUAAAGUAGAAAGCACUACU 65 155094 164 hsa-miR-580-5p UAAUGAUUCAUCAGACUCAGAU 65 156626 165 hsa-miR-6738-3p CUUCUGCCUGCAUUCUACUCCCAG 65 154918 166 hsa-miR-3192-5p UCUGGGAGGUUGUAGCAGUGGAA 65 156212 167 hsa-miR-1277-5p AAAUAUAUAUAUAUAUGUACGUAU 64 154963 168 hsa-miR-196a-3p CGGCAACAAGAAACUGCCUGAG 64 155420 169 hsa-miR-4727-5p AUCUGCCAGCUUCCACAGUGG 63 156069 170 hsa-miR-31-3p UGCUAUGCCAACAUAUUGCCAU 63 154457 171 hsa-miR-5696 CUCAUUUAAGUAGUCUGAUGCC 63 154537 172 hsa-miR-411-5p UAGUAGACCGUAUAGCGUACG 63 155531 173 hsa-miR-329-5p GAGGUUUUCUGGGUUUCUGUUUC 62 154749 174 hsa-miR-6715b-3p CUCAAACCGGCUGUGCCUGUGG 62 156968 175 hsa-miR-3606-5p UUAGUGAAGGCUAUUUUAAUU 62 155152 176 hsa-miR-5692a CAAAUAAUACCACAGUGGGUGU 62 154758 177 hsa-miR-6731-5p UGGGAGAGCAGGGUAUUGUGGA 62 155942 178 hsa-miR-8085 UGGGAGAGAGGACUGUGAGGC 62 155942 179 hsa-miR-3123 CAGAGAAUUGUUUAAUC 61 155351 180 hsa-let-7c-3p CUGUACAACCUUCUAGCUUUCC 61 155428 181 hsa-miR-6083 CUUAUAUCAGAGGCUGUGGG 61 156955 182 hsa-miR-29b-2-5p CUGGUUUCACAUGGUGGCUUAG 61 154366 183 hsa-miR-1185-2-3p AUAUACAGGGGGAGACUCUCAU 60 156452 184 hsa-let-7f-2-3p CUAUACAGUCUACUGUCUUUCC 60 156452 185 hsa-miR-6074 GAUAUUCAGAGGCUAGGUGG 60 154610 186 hsa-miR-1185-1-3p AUAUACAGGGGGAGACUCUUAU 60 156452 187 hsa-miR-4719 UCACAAAUCUAUAAUAUGCAGG 59 157266 188 hsa-miR-7977 UUCCCAGCCAACGCACCA 59 156252 189 hsa-miR-7849-3p GACAAUUGUUGAUCUUGGGCCU 59 155416 190 hsa-miR-4698 UCAAAAUGUAGAGGAAGACCCCA 59 154559 191 hsa-miR-551b-5p GAAAUCAAGCGUGGGUGAGACC 58 154680 192 hsa-miR-2355-5p AUCCCCAGAUACAAUGGACAA 58 157084 193 hsa-miR-548an AAAAGGCAUUGUGGUUUUUG 58 154948 194 hsa-miR-6882-5p UACAAGUCAGGAGCUGAAGCAG 58 157231 195 hsa-miR-3159 UAGGAUUACAAGUGUCGGCCAC 57 155314 196 hsa-miR-4307 AAUGUUUUUUCCUGUUUCC 57 157040 197 hsa-miR-105-5p UCAAAUGCUCAGACUCCUGUGGU 57 154567 198 hsa-miR-7853-5p UCAAAUGCAGAUCCUGACUUC 57 154567 199 hsa-miR-6507-3p CAAAGUCCUUCCUAUUUUUCCC 57 154507 200 hsa-miR-1468-3p AGCAAAAUAAGCAAAUGGAAAA 56 154770 201 hsa-miR-4680-3p UCUGAAUUGUAAGAGUUGUUA 55 156075 202 hsa-miR-6871-5p CAUGGGAGUUCGGGGUGGUUGC 55 155943 203 hsa-miR-7159-3p UUUCUAUGUUAGUUGGAAG 55 154924 204 hsa-miR-6878-5p AGGGAGAAAGCUAGAAGCUGAAG 55 155942 205 hsa-miR-6825-5p UGGGGAGGUGUGGAGUCAGCAU 55 156667 206 hsa-miR-1537-5p AGCUGUAAUUAGUCAGUUUUCU 54 155780 207 hsa-miR-548e-5p CAAAAGCAAUCGCGGUUUUUGC 54 155255 208 hsa-miR-888-5p UACUCAAAAAGCUGUCAGUCA 53 155549 209 hsa-miR-8069 GGAUGGUUGGGGGCGGUCGGCGU 53 156488 210 hsa-miR-4519 CAGCAGUGCGCAGGGCUG 52 155304 211 hsa-miR-6745 UGGGUGGAAGAAGGUCUGGUU 52 154878 212 hsa-miR-196a-1-3p CAACAACAUUAAACCACCCGA 52 155852 213 hsa-miR-7856-5p UUUUAAGGACACUGAGGGAUC 52 155342 214 hsa-miR-892c-5p UAUUCAGAAAGGUGCCAGUCA 52 154428 215 hsa-miR-9-5p UCUUUGGUUAUCUAGCUGUAUGA 52 155194 216 hsa-miR-4795-3p AUAUUAUUAGCCACUUCUGGAU 52 157240 217 hsa-miR-3140-3p AGCUUUUGGGAAUUCAGGUAGU 51 154914 218 hsa-miR-8063 UCAAAAUCAGGAGUCGGGGCUU 51 154559 219 hsa-miR-6792-5p GUAAGCAGGGGCUCUGGGUGA 50 155306

TABLE 11 miRNA modulators targeting SEQ ID NO: 1 Example seed location with reference to Target SEQ ID No Name miRNA Sequence Score NO: 1 1 hsa-miR-6128 ACUGGAAUUGGAGUCAAAA 99 137919 2 hsa-miR-32-5p UAUUGCACAUUACUAAGUUGCA 98 138234 3 hsa-miR-92a-3p UAUUGCACUUGUCCCGGCCUGU 98 138234 4 hsa-miR-92b-3p UAUUGCACUCGUCCCGGCCUCC 98 138234 5 hsa-miR-141-3p UAACACUGUCUGGUAAAGAUGG 97 140276 6 hsa-miR-363-3p AAUUGCACGGUAUCCAUCUGUA 97 138234 7 hsa-miR-200a-3p UAACACUGUCUGGUAACGAUGU 97 140276 8 hsa-miR-367-3p AAUUGCACUUUAGCAAUGGUGA 97 138234 9 hsa-miR-25-3p CAUUGCACUUGUCUCGGUCUGA 97 138234 10 hsa-miR-30d-5p UGUAAACAUCCCCGACUGGAAG 94 139356 11 hsa-miR-30c-5p UGUAAACAUCCUACACUCUCAGC 94 139356 12 hsa-miR-30b-5p UGUAAACAUCCUACACUCAGCU 94 139356 13 hsa-miR-221-3p AGCUACAUUGUCUGCUGGGUUUC 94 138255 14 hsa-miR-222-3p AGCUACAUCUGGCUACUGGGU 94 138255 15 hsa-miR-511-3p AAUGUGUAGCAAAAGACAGA 94 138650 16 hsa-miR-30a-5p UGUAAACAUCCUCGACUGGAAG 94 139356 17 hsa-miR-30e-5p UGUAAACAUCCUUGACUGGAAG 94 139356 18 hsa-miR-6875-3p AUUCUUCCUGCCCUGGCUCCAU 92 137572 19 hsa-miR-2052 UGUUUUGAUAACAGUAAUGU 91 138624 20 hsa-miR-510-3p AUUGAAACCUCUAAGAGUGGA 91 140413 21 hsa-miR-3163 UAUAAAAUGAGGGCAGUAAGAC 91 138581 22 hsa-miR-203b-3p UUGAACUGUUAAGAACCACUGGA 91 138514 23 hsa-miR-6806-5p UGUAGGCAUGAGGCAGGGCCCAGG 90 139700 24 hsa-miR-500a-3p AUGCACCUGGGCAAGGAUUCUG 90 138233 25 hsa-miR-6796-3p GAAGCUCUCCCCUCCCCGCAG 90 138338 26 hsa-miR-3140-3p AGCUUUUGGGAAUUCAGGUAGU 89 137629 27 hsa-miR-4262 GACAUUCAGACUACCUG 88 140173 28 hsa-miR-4494 CCAGACUGUGGCUGACCAGAGG 88 138304 29 hsa-miR-767-5p UGCACCAUGGUUGUCUGAGCAUG 87 138232 30 hsa-miR-9-5p UCUUUGGUUAUCUAGCUGUAUGA 86 138373 31 hsa-miR-12136 GAAAAAGUCAUGGAGGCC 86 138037 32 hsa-miR-4705 UCAAUCACUUGGUAAUUGCUGU 86 137742 33 hsa-miR-570-3p CGAAAACAGCAAUUACCUUUGC 86 138164 34 hsa-miR-4659b-3p UUUCUUCUUAGACAUGGCAGCU 85 137573 35 hsa-miR-181d-5p AACAUUCAUUGUUGUCGGUGGGU 85 140173 36 hsa-miR-4659a-3p UUUCUUCUUAGACAUGGCAACG 85 137573 37 hsa-miR-181b-5p AACAUUCAUUGCUGUCGGUGGGU 85 140173 38 hsa-miR-8054 GAAAGUACAGAUCGGAUGGGU 85 138469 39 hsa-miR-548av-5p AAAAGUACUUGCGGAUUU 85 138469 40 hsa-miR-181c-5p AACAUUCAACCUGUCGGUGAGU 85 140173 41 hsa-miR-181a-5p AACAUUCAACGCUGUCGGUGAGU 85 140173 42 hsa-miR-548k AAAAGUACUUGCGGAUUUUGCU 85 138469 43 hsa-miR-4795-3p AUAUUAUUAGCCACUUCUGGAU 84 140564 44 hsa-miR-548ar-3p UAAAACUGCAGUUAUUUUUGC 84 138993 45 hsa-miR-12114 CAGGUGGAGGUGUGAGGUC 84 137663 46 hsa-miR-105-5p UCAAAUGCUCAGACUCCUGUGGU 83 137736 47 hsa-miR-4500 UGAGGUAGUAGUUUCUU 83 139977 48 hsa-miR-7853-5p UCAAAUGCAGAUCCUGACUUC 83 137736 49 hsa-miR-1231 GUGUCUGGGCGGACAGCUGC 83 138637 50 hsa-let-7e-5p UGAGGUAGGAGGUUGUAUAGUU 82 139977 51 hsa-miR-98-5p UGAGGUAGUAAGUUGUAUUGUU 82 139977 52 hsa-let-7f-5p UGAGGUAGUAGAUUGUAUAGUU 82 139977 53 hsa-let-7c-5p UGAGGUAGUAGGUUGUAUGGUU 82 139977 54 hsa-let-7i-5p UGAGGUAGUAGUUUGUGCUGUU 82 139977 55 hsa-let-7b-5p UGAGGUAGUAGGUUGUGUGGUU 82 139977 56 hsa-let-7g-5p UGAGGUAGUAGUUUGUACAGUU 82 139977 57 hsa-let-7a-5p UGAGGUAGUAGGUUGUAUAGUU 82 139977 58 hsa-miR-548au-5p AAAAGUAAUUGCGGUUUUUGC 81 138468 59 hsa-miR-548y AAAAGUAAUCACUGUUUUUGCC 81 138468 60 hsa-miR-548az-3p AAAAACUGCAAUCACUUUUGC 81 138993 61 hsa-miR-548a-5p AAAAGUAAUUGCGAGUUUUACC 81 138468 62 hsa-miR-548ae-5p AAAAGUAAUUGUGGUUUUUG 81 138468 63 hsa-miR-548ab AAAAGUAAUUGUGGAUUUUGCU 81 138468 64 hsa-miR-548am-5p AAAAGUAAUUGCGGUUUUUGCC 81 138468 65 hsa-miR-548h-5p AAAAGUAAUCGCGGUUUUUGUC 81 138468 66 hsa-let-7d-5p AGAGGUAGUAGGUUGCAUAGUU 81 139977 67 hsa-miR-548ar-5p AAAAGUAAUUGCAGUUUUUGC 81 138468 68 hsa-miR-548ap-5p AAAAGUAAUUGCGGUCUUU 81 138468 69 hsa-miR-548a-3p CAAAACUGGCAAUUACUUUUGC 81 138993 70 hsa-miR-548ak AAAAGUAACUGCGGUUUUUGA 81 138468 71 hsa-miR-548bc AAAAACUGUGAUUACUUUUGC 81 138993 72 hsa-miR-548w AAAAGUAACUGCGGUUUUUGCCU 81 138468 73 hsa-miR-548f-3p AAAAACUGUAAUUACUUUU 81 138993 74 hsa-miR-548ay-5p AAAAGUAAUUGUGGUUUUUGC 81 138468 75 hsa-miR-548o-5p AAAAGUAAUUGCGGUUUUUGCC 81 138468 76 hsa-miR-548i AAAAGUAAUUGCGGAUUUUGCC 81 138468 77 hsa-miR-548p UAGCAAAAACUGCAGUUACUUU 81 137683 78 hsa-miR-548as-5p AAAAGUAAUUGCGGGUUUUGCC 81 138468 79 hsa-miR-548ad-5p AAAAGUAAUUGUGGUUUUUG 81 138468 80 hsa-miR-8087 GAAGACUUCUUGGAUUACAGGGG 81 140023 81 hsa-miR-548aq-5p GAAAGUAAUUGCUGUUUUUGCC 81 138468 82 hsa-miR-548e-3p AAAAACUGAGACUACUUUUGCA 81 138993 83 hsa-miR-548bb-5p AAAAGUAACUAUGGUUUUUGCC 81 138468 84 hsa-miR-4458 AGAGGUAGGUGUGGAAGAA 81 139977 85 hsa-miR-548j-5p AAAAGUAAUUGCGGUCUUUGGU 81 138468 86 hsa-miR-559 UAAAGUAAAUAUGCACCAAAA 81 138468 87 hsa-miR-548c-5p AAAAGUAAUUGCGGUUUUUGCC 81 138468 88 hsa-miR-548d-5p AAAAGUAAUUGUGGUUUUUGCC 81 138468 89 hsa-miR-548b-5p AAAAGUAAUUGUGGUUUUGGCC 81 138468 90 hsa-miR-4503 UUUAAGCAGGAAAUAGAAUUUA 81 140841 91 hsa-miR-6505-5p UUGGAAUAGGGGAUAUCUCAGC 81 137918 92 hsa-miR-1250-3p ACAUUUUCCAGCCCAUUCA 80 138672 93 hsa-miR-4656 UGGGCUGAGGGCAGGAGGCCUGU 80 139534 94 hsa-miR-2114-5p UAGUCCCUUCCUUGAAGCGGUC 79 138976 95 hsa-miR-5692a CAAAUAAUACCACAGUGGGUGU 79 140991 96 hsa-miR-187-5p GGCUACAACACAGGACCCGGGC 79 138286 97 hsa-miR-9983-3p UUUUUUGCUGGAACAUUUCUGG 79 138694 98 hsa-miR-1255b-2-3p AACCACUUUCUUUGCUCAUCCA 79 140852 99 hsa-miR-4469 GCUCCCUCUAGGGUCGCUCGGA 79 140433 100 hsa-miR-129-5p CUUUUUGCGGUCUGGGCUUGC 78 138694 101 hsa-miR-495-5p GAAGUUGCCCAUGUUAUUUUCG 78 140388 102 hsa-miR-302c-5p UUUAACAUGGGGGUACCUGCUG 77 140279 103 hsa-miR-23a-3p AUCACAUUGCCAGGGAUUUCC 76 140696 104 hsa-miR-486-5p UCCUGUACUGAGCUGCCCCGAG 76 140295 105 hsa-miR-5694 CAGAUCAUGGGACUGUCUCAG 76 138570 106 hsa-miR-23b-3p AUCACAUUGCCAGGGAUUACCAC 76 140696 107 hsa-miR-584-5p UUAUGGUUUGCCUGGGACUGAG 76 140404 108 hsa-miR-23c AUCACAUUGCCAGUGAUUACCC 76 140696 109 hsa-miR-3160-5p GGCUUUCUAGUCUCAGCUCUCC 76 138059 110 hsa-miR-489-3p GUGACAUCACAUAUACGGCAGC 75 137624 111 hsa-miR-548g-3p AAAACUGUAAUUACUUUUGUAC 75 138029 112 hsa-miR-7162-3p UCUGAGGUGGAACAGCAGC 75 139979 113 hsa-miR-3145-5p AACUCCAAACACUCAAAACUCA 74 138179 114 hsa-miR-520e-5p CUCAAGAUGGAAGCAGUUUCUG 74 140818 115 hsa-miR-302b-3p UAAGUGCUUCCAUGUUUUAGUAG 73 138857 116 hsa-miR-302d-3p UAAGUGCUUCCAUGUUUGAGUGU 73 138857 117 hsa-miR-520f-5p CCUCUAAAGGGAAGCGCUUUCU 73 138335 118 hsa-miR-6885-3p CUUUGCUUCCUGCUCCCCUAG 73 138053 119 hsa-miR-302a-3p UAAGUGCUUCCAUGUUUUGGUGA 73 138857 120 hsa-miR-302e UAAGUGCUUCCAUGCUU 73 138857 121 hsa-miR-302c-3p UAAGUGCUUCCAUGUUUCAGUGG 73 138857 122 hsa-miR-4719 UCACAAAUCUAUAAUAUGCAGG 73 137738 123 hsa-miR-4252 GGCCACUGAGUCAGCACCA 73 138088 124 hsa-miR-5582-3p UAAAACUUUAAGUGUGCCUAGG 72 139763 125 hsa-miR-577 UAGAUAAAAUAUUGGUACCUG 72 141041 126 hsa-miR-6809-3p CUUCUCUUCUCUCCUUCCCAG 72 140905 127 hsa-miR-543 AAACAUUCGCGGUGCACUUCUU 72 139720 128 hsa-miR-3927-5p GCCUAUCACAUAUCUGCCUGU 72 140914 129 hsa-miR-580-3p UUGAGAAUGAUGAAUCAUUAGG 71 137588 130 hsa-miR-95-5p UCAAUAAAUGUCUGUUGAAUU 71 140714 131 hsa-miR-548c-3p CAAAAAUCUCAAUUACUUUUGC 71 138464 132 hsa-miR-4775 UUAAUUUUUUGUUUCGGUCACU 71 140417 133 hsa-miR-4307 AAUGUUUUUUCCUGUUUCC 70 137815 134 hsa-miR-6828-3p AUCUGCUCUCUUGUUCCCAG 70 140091 135 hsa-miR-541-5p AAAGGAUUCUGCUGUCGGUCCCACU 70 138325 136 hsa-miR-4766-5p UCUGAAAGAGCAGUUGGUGUU 69 138194 137 hsa-miR-499a-5p UUAAGACUUGCAGUGAUGUUU 68 139774 138 hsa-miR-6748-3p UCCUGUCCCUGUCUCCUACAG 68 138822 139 hsa-miR-6844 UUCUUUGUUUUUAAUUCACAG 67 137786 140 hsa-miR-651-3p AAAGGAAAGUGUAUCCUAAAAG 67 138438 141 hsa-miR-605-3p AGAAGGCACUAUGAGAUUUAGA 67 138600 142 hsa-miR-29a-5p ACUGAUUUCUUUUGGUGUUCAG 66 137999 143 hsa-miR-4667-3p UCCCUCCUUCUGUCCCCACAG 65 140432 144 hsa-miR-652-5p CAACCCUAGGAGAGGGUGCCAUUCA 65 140004 145 hsa-miR-6513-5p UUUGGGAUUGACGCCACAUGUCU 65 140584 146 hsa-miR-137-3p UUAUUGCUUAAGAAUACGCGUAG 65 138236 147 hsa-miR-4289 GCAUUGUGCAGGGCUAUCA 64 140208 148 hsa-miR-3180-5p CUUCCAGACGCUCCGCCCCACGUCG 64 140878 149 hsa-miR-506-3p UAAGGCACCCUUCUGAGUAGA 64 138599 150 hsa-miR-1470 GCCCUCCGCCCGUGCACCCCG 64 140432 151 hsa-miR-6867-3p CUCUCCCUCUUUACCCACUAG 64 140434 152 hsa-miR-124-3p UAAGGCACGCGGUGAAUGCCAA 64 138599 153 hsa-miR-627-3p UCUUUUCUUUGAGACUCACU 64 139966 154 hsa-miR-3613-3p ACAAAAAAAAAAGCCCAACCCUUC 63 140927 155 hsa-miR-1283 UCUACAAAGGAAAGCGCUUUCU 63 138285 156 hsa-miR-5582-5p UAGGCACACUUAAAGUUAUAGC 63 138598 157 hsa-miR-369-3p AAUAAUACAUGGUUGAUCUUU 62 140989 158 hsa-miR-548av-3p AAAACUGCAGUUACUUUUGC 62 138745 159 hsa-miR-3146 CAUGCUAGGAUAGAAAGAAUGG 62 139155 160 hsa-let-7c-3p CUGUACAACCUUCUAGCUUUCC 62 140293 161 hsa-miR-520a-3p AAAGUGCUUCCCUUUGGACUGU 61 138857 162 hsa-miR-520e-3p AAAGUGCUUCCUUUUUGAGGG 61 138857 163 hsa-miR-520d-3p AAAGUGCUUCUCUUUGGUGGGU 61 138857 164 hsa-miR-520b-3p AAAGUGCUUCCUUUUAGAGGG 61 138857 165 hsa-miR-629-3p GUUCUCCCAACGUAAGCCCAGC 61 137601 166 hsa-miR-520c-3p AAAGUGCUUCCUUUUAGAGGGU 61 138857 167 hsa-miR-373-3p GAAGUGCUUCGAUUUUGGGGUGU 61 138857 168 hsa-miR-372-3p AAAGUGCUGCGACAUUUGAGCGU 61 138857 169 hsa-miR-20a-5p UAAAGUGCUUAUAGUGCAGGUAG 60 137756 170 hsa-miR-106b-5p UAAAGUGCUGACAGUGCAGAU 60 137756 171 hsa-miR-519d-3p CAAAGUGCCUCCCUUUAGAGUG 60 137756 172 hsa-miR-93-5p CAAAGUGCUGUUCGUGCAGGUAG 60 137756 173 hsa-miR-17-5p CAAAGUGCUUACAGUGCAGGUAG 60 137756 174 hsa-miR-188-3p CUCCCACAUGCAGGGUUUGCA 60 138494 175 hsa-miR-7-1-3p CAACAAAUCACAGUCUGCCAUA 60 137767 176 hsa-miR-526b-3p GAAAGUGCUUCCUUUUAGAGGC 60 137756 177 hsa-miR-20b-5p CAAAGUGCUCAUAGUGCAGGUAG 60 137756 178 hsa-miR-7-2-3p CAACAAAUCCCAGUCUACCUAA 60 137767 179 hsa-miR-106a-5p AAAAGUGCUUACAGUGCAGGUAG 60 137756 180 hsa-miR-520a-5p CUCCAGAGGGAAGUACUUUCU 59 140878 181 hsa-miR-3202 UGGAAGGGAGAAGAGCUUUAAU 59 138897 182 hsa-miR-10522-5p AGAAGAAUUGGCCUACUCAGG 59 140010 183 hsa-miR-3185 AGAAGAAGGCGGUCGGUCUGCGG 59 140010 184 hsa-miR-3154 CAGAAGGGGAGUUGGGAGCAGA 59 138602 185 hsa-miR-647 GUGGCUGCACUCACUUCCUUC 59 138202 186 hsa-miR-525-5p CUCCAGAGGGAUGCACUUUCU 59 140878 187 hsa-miR-5695 ACUCCAAGAAGAAUCUAGACAG 59 138178 188 hsa-miR-511-5p GUGUCUUUUGCUCUGCAGUCA 58 137876 189 hsa-miR-4287 UCUCCCUUGAGGGCACUUU 58 140434 190 hsa-miR-3925-3p ACUCCAGUUUUAGUUCUCUUG 58 137964 191 hsa-miR-513c-3p UAAAUUUCACCUUUCUGAGAAGA 58 139561 192 hsa-miR-6079 UUGGAAGCUUGGACCAACUAGCUG 58 137935 193 hsa-miR-4685-3p UCUCCCUUCCUGCCCUGGCUAG 58 140434 194 hsa-miR-513a-3p UAAAUUUCACCUUUCUGAGAAGG 58 139561 195 hsa-miR-548v AGCUACAGUUACUUUUGCACCA 57 138287 196 hsa-miR-451b UAGCAAGAGAACCAUUACCAUU 57 140056 197 hsa-miR-5680 GAGAAAUGCUGGACUAAUCUGC 57 138170 198 hsa-miR-3120-3p CACAGCAAGUGUAGACAGGCA 57 137685 199 hsa-miR-6831-5p UAGGUAGAGUGUGAGGAGGAGGUC 56 139976 200 hsa-miR-3662 GAAAAUGAUGAGUAGUGACUGAUG 56 139088 201 hsa-miR-5584-5p CAGGGAAAUGGGAAGAACUAGA 56 138966 202 hsa-miR-3927-3p CAGGUAGAUAUUUGAUAGGCAU 56 139976 203 hsa-miR-7113-3p CCUCCCUGCCCGCCUCUCUGCAG 56 140434 204 hsa-miR-520f-3p AAGUGCUUCCUUUUAGAGGGUU 56 137821 205 hsa-miR-3606-3p AAAAUUUCUUUCACUACUUAG 55 139561 206 hsa-miR-424-3p CAAAACGUGAGGCGCUGCUAU 55 140194 207 hsa-miR-10523-5p GACAAUGAUGAGAAGACCUGAGGA 55 138654 208 hsa-miR-3664-3p UCUCAGGAGUAAAGACAGAGUU 55 139554 209 hsa-miR-6728-5p UUGGGAUGGUAGGACCAGAGGGG 55 140360 210 hsa-miR-27b-5p AGAGCUUAGCUGAUUGGUGAAC 54 137550 211 hsa-miR-584-3p UCAGUUCCAGGCCAACCAGGCU 54 140368 212 hsa-miR-4711-5p UGCAUCAGGCCAGAAGACAUGAG 54 137696 213 hsa-miR-4511 GAAGAACUGUUGCAUUUGCCCU 54 137561 214 hsa-miR-224-5p UCAAGUCACUAGUGGUUCCGUUUAG 54 137885 215 hsa-miR-5089-5p GUGGGAUUUCUGAGUAGCAUC 53 138267 216 hsa-miR-7973 UGUGACCCUAGAAUAAUUAC 53 138025 217 hsa-miR-4473 CUAGUGCUCUCCGUUACAAGUA 53 137822 218 hsa-miR-3159 UAGGAUUACAAGUGUCGGCCAC 53 139684 219 hsa-miR-4764-5p UGGAUGUGGAAGGAGUUAUCU 53 138778 220 hsa-miR-664b-3p UUCAUUUGCCUCCCAGCCUACA 52 138455 221 hsa-miR-1266-5p CCUCAGGGCUGUAGAACAGGGCU 52 139554 222 hsa-miR-202-3p AGAGGUAUAGGGCAUGGGAA 52 139978 223 hsa-miR-499b-5p ACAGACUUGCUGUGAUGUUCA 52 138305 224 hsa-miR-4518 GCUCAGGGAUGAUAACUGUGCUGAGA 52 139554 225 hsa-miR-579-3p UUCAUUUGGUAUAAACCGCGAUU 52 138455 226 hsa-miR-1264 CAAGUCUUAUUUGAGCACCUGUU 52 137710 227 hsa-miR-6817-3p UCUCUCUGACUCCAUGGCA 52 139220 228 hsa-miR-148b-5p AAGUUCUGUUAUACACUCAGGC 52 139016 229 hsa-miR-6083 CUUAUAUCAGAGGCUGUGGG 51 138730 230 hsa-miR-19b-2-5p AGUUUUGCAGGUUUGCAUUUCA 51 138625 231 hsa-miR-19b-1-5p AGUUUUGCAGGUUUGCAUCCAGC 51 138625 232 hsa-miR-4666a-5p AUACAUGUCAGAUUGUAUGCC 51 138120 233 hsa-miR-3680-3p UUUUGCAUGACCCUGGGAGUAGG 51 138692 234 hsa-miR-12124 GAGGAAAUGCAGAUGCUGGA 51 138678 235 hsa-miR-19a-5p AGUUUUGCAUAGUUGCACUACA 51 138625 236 hsa-miR-545-3p UCAGCAAACAUUUAUUGUGUGC 51 137684 237 hsa-miR-4668-3p GAAAAUCCUUUUUGUUUUUCCAG 51 140502 238 hsa-miR-4279 CUCUCCUCCCGGCUUC 51 137600 239 hsa-miR-6124 GGGAAAAGGAAGGGGGAGGA 51 140061 240 hsa-miR-4709-5p ACAACAGUGACUUGCUCUCCAA 51 140032 241 hsa-miR-6134 UGAGGUGGUAGGAUGUAGA 50 139927

TABLE 12 Small molecule modulators targeting FNIPI Exemplar Dissociation No Constants Exemplar SMILES Exemplar Image Scaffold SMILES Scaffold Image  1 5.087e-09 O═C1CC(/C═C/CCN2CCC(c3 ccc(Cl)cc3)═CC2)C2C[C@@] 23C═C(CC═C3Cl)N1

O═C1CC(C═CCCN2CC═ C(c3ccccc3)CC2)C2C C23C═CCC(═C3)N1

 2 3.017e-08 [N]c1c2c(c3c(c1C14CCCC1C 4)CNC3═O)-c1ccccc1[N]2

O═C1NCc2c(C34CCCC 3C4)cc3c(c21)- c1ccccc1[N]3

 3 3.547e-08 O═C(NCc1ccc(OC(F)(F)F)cc1) C1C2C═CCCC2C(═O)N1C1 CCOCC1

O═C(NCc1ccccc1)C1C2 C═CCCC2C(═O)N1C1C COCC1

 4 7.967e-08 O═C1NC(═O)C2C1C1C3CC═ CC[C@]34N3C5CCC(O5)n5c (c2c2ccccc25)[C@]134

O═C1NC(═O)C2C1c1c3 n(c4ccccc14)C1CCC(O1) N1C45CC═CCC4C2C3 15

 5 7.967e-08 CC1CC(O)[C@]2(F)C(CCC3 OC4(CCCC4)O[C@]13C(═O) COC(C)═O)CCC1═CC(═O)C═ C[C@@]12C

O═C1C═CC2C(═C1)CC C1CCC3OC4(CCCC4)O C3CCCC12

 6 9.367e-08 COC1CCC2C(CCN(C)C2CC2 CCC(OC3C[C@]4(OC)C(CC5 C/C(═C\C4OC)CCN5C)C═CC 3OC(═O)c3ccc([N+](═O)[O−])c c3)C═C2)C1

O═C(OC1C═CC2CC3C C(═CCC2CC1OC1C═C C(CC2NCCC4CCCCC4 2)CC1)CCN3)c1ccccc1

 7 9.367e-08 Cc1c(NC(═O)c2cc3c(s2)CCC C3)cccc1- c1cn(C)c(═O)c(Nc2ccc(C3C(═ O)N(C)CCN3C)cc2)n1

O═C(Nc1cccc(- c2c[nH]c(═O)c(Nc3ccc(C 4NCCNC4═O)cc3)n2)c1) c1cc2c(s1)CCCC2

 8 1.101e-07 COc1ccc(C2([O])[N]C2C2C[C @]23CC═CC(C2═NNC(═O)S C2C)═C3)cc1OC

O═C1NN═C(C2═CC3(C C═C2)CC3C2[N]C2c2cc ccc2)CS1

 9 1.101e-07 CC(C(═O)Nc1cc(C)ccn1)c1cc cc(C(═O)c2ccccc2)c1

O═C(Cc1cccc(C(═O)c2c cccc2)c1)Nc1ccccn1

 10 1.101e-07 CNC1CC(N)C(O)C(OC2OCC (O)C3O[C@]4(OC(CN)C(O)C (O)C4O)OC23)C1O

C1CCC(OC2OCCC3OC 4(CCCCO4)OC32)CC1

 11 1.295e-07 [N]C(C[O])CCC(O)[C@]12CC 1CC(Cl)═C2

C1═CC2CC2C1

 12 1.522e-07 Cn1cc(C2═C(c3cn(CCCSC(═ N)N)c4ccccc34)C(═O)NC2═O) c2ccccc21

O═C1NC(═O)C(c2c[nH]c 3ccccc23)═C1c1c[nH]c2 ccccc12

 13 1.522e-07 Cc1cn2nc(C3═CC(═O)N4CC (N5CCNC6(CC6)C5)═CC═C4 [N]3)cc(C)c2n1

O═C1C═C(c2ccc3nccn3 n2)[N]C2═CC═C(N3CCN C4(CC4)C3)CN12

 14 1.522e-07 O═c1c(- c2ccc(O)cc2)coc2c(C3OC(CO) C(O)C(O)C3O)c(O)ccc12

O═c1c(- c2ccccc2)coc2c(C3CCC CO3)cccc12

 15 1.789e-07 CCOc1ccc(N2C([O])C3CCC═ NC3═NC2C(C)N(Cc2cccnc2) C(═O)Cc2ccc(OC(F)(F)F)cc2) cc1

O═C(Cc1ccccc1)N(Cc1c ccnc1)CC1N═C2N═CCC C2CN1c1ccccc1

 16 2.104e-07 COc1ccc2c(c1)C(C(═O)N(Cc1 ccc(N(C)C)cc1)c1ccc(C(C)C)c c1)CCC2

c1ccc2c(c1)CCCC2

 17 2.473e-07 Cc1c(C)c2c(c(C)c1O)CC[C@] (C)(COc1ccc(CC3SC(═O)NC 3═O)cc1)O2

c1ccc2c(c1)CCCO2

 18 2.908e-07 O═C(Nc1ccc(F)cc1)N1CCc2c cccc2C1c1ccc(C(F)(F)F)cc1

c1ccc(C2NCCc3ccccc32) cc1

 19 2.908e-07 Oc1cc2c(cc1O)C(CC1CC1Cc 1ccccc1)NCC2

c1ccc2c(c1)CCNC2

 20 3.419e-07 CC1CC(O)C2C(CCC3CC(═O)O) CC[C@]32C)C1CCC(O)C(═O) COC(═O)CC(C)(C)C

C1CCC2CCCCC2C1

 21 4.019e-07 CCOC(═O)C(C)Oc1cccc2c1C CN(CC(═O)Nc1ccc3c(c1)OC CO3)C2═O

c1ccc2c(c1)OCCO2

 22 4.725e-07 O═C(/C═C/c1ccccc1)NC(NC(═ S)Nc1cccc2cccnc12)C(Cl)(Cl) Cl

c1ccc2ncccc2c1

 23 4.725e-07 CCOc1nc2cccc(C(═O)OC(C)═ O)c2n1Cc1ccc(- c2ccccc2C2═N[N]N[N]2)cc1

c1ccc2[nH]cnc2c1

 24 4.725e-07 CC1═C(C(═O)Nc2cc3c[nH]nc 3cc2F)C(c2ccc(C(F)(F)F)cc2) CC(═O)N1

c1ccc2n[nH]cc2c1

 25 4.725e-07 C(═N/n1cnnc1)\c1ccc(- c2ccccc2)cc1

c1ccc(-c2ccccc2)cc1

 26 4.725e-07 [O]C(═O)C1Cc2ccccc2C2═C C(═O)CCC12

O═C1C═C2c3ccccc3CC C2CC1

 27 4.725e-07 NC1NN═CC(c2c(O)ccc3ccccc 23)N1

c1ccc2ccccc2c1

 28 4.725e-07 c1ccc(- c2cc3c(ccc4nonc43)nn2)cc1

c1ccc2nonc2c1

 29 5.555e-07 CN1CCCCC1CN1CC(C(═O)c 2cc([N+](═O)[O−])ccc21)c2ccc cc21

c1ccc2c(c1)CCN2

 30 5.555e-07 Cn1c(COc2ccc(CC3SC(═O)N C3═O)cc2)nc2ccccc2c1═O

O═c1[nH]cnc2ccccc12

 31 5.555e-07 c1ccc(- c2nn3nnnc3c3ccccc23)cc1

c1cnn2nnnc2c1

 32 6.531e-07 CC(C)Oc1ccc(-c2nc(- c3cccc4c3CCC4NCCC(═O)O) no2)cc1C#N

c1ccc2c(c1)CCC2

 33 6.531e-07 Cc1ccc(- c2ccc3c(c2)C(N)C(═O)N3)cc1

O═C1Cc2ccccc2N1

 34 6.531e-07 [NH]CCc1cc(O)c(O)cc1CCc1c cc(O)cc1

c1ccc(CCc2ccccc2)cc1

 35 6.531e-07 Nc1cc(-c2ccc(- c3cc[nH]n3)cc2)ccn1

c1ccc(-c2cc[nH]n2)cc1

 36 6.531e-07 [O][C@@]12C3C4CC[C@]56 C4C4[C@@]31C2C1CC(═O) C5[C@@]146

C1CC2CCC2C1

 37 6.531e-07 O═C1C2C3CCC(C3)C2C(═O) c2ccccc21

C1CC2CCC1C2

 38 6.531e-07 CN1CCCC(n2nc(Cc3ccc(Cl)c c3)c3ccccc3c2═O)CC1

O═c1[nH]ncc2ccccc12

 39 6.531e-07 c1ccc(C2N═N[C@@]3(NN2)c 2cccnc23)nc1

c1ccc(C2N═NC3(NN2)c 2cccnc23)nc1

 40 6.531e-07 NC1═N[N]c2c(c3ccccc3c3ccc cc23)[N]1

C1═N[N]c2ccccc2[N]1

 41 7.679e-07 O═C1c2ccccc2C(═O)c2ccccc 21

O═C1c2ccccc2C(═O)c2c cccc21

 42 7.679e-07 OC1C2C(C(O)c3ccccc32)c2c cccc21

c1ccc2c(c1)CC1c3ccccc 3CC21

 43 7.679e-07 C1═CC(c2ccc3ccccc3c2)N2N═ C[N]C2═N1

C1═CN═C2[N]C═NN2C1

 44 7.679e-07 Cc1ccc(- c2ccc3c(c2)C(═O)C(═O)N3)cc 1

O═C1Nc2ccc (c3ccccc3)cc2C1═O

 45 7.679e-07 CC1C2C(C(C═C(C)C)C═C1C) C(═O)NNC2═O

O═C1NNC(═O)C2CC═C CC12

 46 7.679e-07 Cc1cc(NC(═O)C2CCc3nc[nH] c3C2)no1

c1nc2c([nH]1)CCCC2

 47 7.679e-07 NNc1nc2c(- c3ccccc3)c[nH]c2c(═O)[nH]1

O═c1[nH]cnc2c(- c3ccccc3)c[nH]c12

 48 7.679e-07 OC1C(O)c2ccccc2-c2ccccc21

c1ccc2c(c1)CCc1ccccc1- 2

 49 7.679e-07 Nc1nnc(-c2ccc(- c3ccccc3)cc2)o1

c1ccc(-c2nnco2)cc1

 50 7.679e-07 CC1═C2N═C(c3cc(F)c4nc(C) cn4c3)CC(═O)N2CC(N2CCN (C)CC2)═C1

c1ccn2ccnc2c1

 51 7.679e-07 Cc1ccccc1C1NC(═O)c2ccccc 2N1

O═C1NC(c2ccccc2)Nc2 ccccc21

 52 7.679e-07 O═S1(═O)C2C═CC═CC2Oc2 ccccc21

O═S1(═O)c2ccccc2OC2 C═CC═CC21

 53 7.679e-07 Cn1c2ccccc2c2cc(CC([O])═O) ccc21

c1ccc2c(c1)[nH]c1ccccc 12

 54 7.679e-07 NC(═O)C12C3C1c1ccccc1C2 c1ccccc13

c1ccc2c(c1)C1c3ccccc3 C3C2C13

 55 9.027e-07 Cc1cc(NC(═O)c2cc3ncccn3n2) no1

c1cnc2ccnn2c1

 56 9.027e-07 [O]C(═O)c1cc (c2ccc3c(c2)CCCC3)on1

c1cc(- c2ccc3c(c2)CCCC3)on1

 57 9.027e-07 C[c@12CCc3ccccc3[C@]1 (C)NC(═O)O2

O═C1NC2c3ccccc3CCC 2O1

 58 9.027e-07 O═C1c2ccccc2- c2(nH]c(═O)c3ccccc3c21

O═c1(nH)ccc2ccccc12

 59 9.027e-07 C1═Cc2nonc2C2C═C(c3cccc c3)N═C12

C1═Cc2nonc2CC1

 60 9.027e-07 c1ccc(- n2nc3ccc4nonc4c3n2)cc1

c1ccc2n[nH]nc2c1

 61 9.027e-07 CC1CCCN(C(═O)c2ccc3[nH]n nc3c2)C1

c1ccc2[nH]nnc2c1

 62 9.027e-07 C1═NC2═NO[N]C2═NC1c1cc 2ccccc2o1

C1═NC2═NO[N]C2═NC1

 63 9.027e-07 OC1CC2CCC3CC(c4ccccc4) C1C23

C1CC2CCCC2C1

 64 9.027e-07 Cc1ccccclC1NC(═O)c2cccnc 2N1

O═C1NC(c2ccccc2)Nc2 ncccc21

 65 9.027e-07 C[C@]1(O)CC(c2ccccc2)c2c ([nH][nH]c2═O)C1

O═c1[nH][nH]c2c1CCCC 2

 66 1.061e-06 c1ccc(-c2n[nH]c(- c3cccnc3)n2)cc1

c1ccc(-c2n[nH]c(- c3cccnc3)n2)cc1

 67 1.061e-06 O═c1[nH]c(- c2cccc(O)c2)nc2ccccc12

O═c1[nH]c (-c2ccccc2)nc2ccccc12

 68 1.061e-06 Cc1cc(- c2cncc(C(C)(C)O)c2)ccc10

c1ccc(-c2cccnc2)cc1

 69 1.061e-06 CC(═O)N1CCCC(c2cc3cccnc 3[nH]2)C1

c1cnc2[nH]ccc2c1

 70 1.061e-06 Cc1ccc(N2Cc3ccccc3C(═O)N 2)cc1

O═C1NN(c2ccccc2)Cc2 ccccc21

 71 1.061e-06 CC1═CCC2C(C1)C(═O)N(c1c c(C)on1)C2═O

O═C1C2CC═CCC2C(═O) N1c1ccon1

 72 1.061e-06 CCC(C(═O)O)c1ccc(N2Cc3cc ccc3C2═O)cc1

O═C1NCc2ccccc21

 73 1.061e-06 c1cc2c3c(cccc3c1)C1═C2[N] C2═NO[N]C2═N1

C1═Cc2cccc3cccc1c23

 74 1.061e-06 CC(═O)CC(c1ccc([N+](═O)[O-]) cc1)c1c(O)c2ccccc2oc1═O

O═c1ccc2ccccc2o1

 75 1.061e-06 Cc1cc(C)c2c(c1)C1CCCC1C (C([O])═O)N2

c1ccc2c(c1)CCCN2

 76 1.061e-06 Cc1cc(Cn2c(═O)ccc3ccccc32) on1

O═c1ccc2ccccc2[nH]1

 77 1.061e-06 CC(NN)c1ccc2c(c1)C═C1CC═ CC═C12

C1═CCC2═Cc3ccccc3C 2═C1

 78 1.061e-06 Cc1ccc(C2CC(═O)c3cn[nH]c3 C2)cc1

O═C1CC(c2ccccc2)Cc2 [nH]ncc21

 79 1.061e-06 O═C1CC(c2nc(- c3cccc(F)c3)no2)CN1

O═C1CC(c2nc(- c3ccccc3)no2)CN1

 80 1.061e-06 O═C1CCC(N2C(═O)c3ccccc3 C2═O)C(═O)N1

O═C1NC(═O)c2ccccc21

 81 1.061e-06 Nc1ccc2c(c1)/C(═N/O)c1cccc c1-2

N═C1c2ccccc2- c2ccccc21

 82 1.061e-06 Cc1cccc(C(═O)n2nnc3ccccc3 2)c1

O═C(c1cccccl)n1nnc2c cccc21

 83 1.248e-06 Cc1ccc2c(ccc3nc(N)nc(N)c32) c1

c1ccc2ncncc2c1

 84 1.248e-06 Oc1cccc(C2N═C(c3ccccn3)[N] N2)c1

c1ccc(C2N═C(c3ccccn3) [N]N2)cc1

 85 1.248e-06 [O]C(═O)c1ccc(- c2ccc3c(c2)OCO3)cc1

c1ccc2c(c1)OCO2

 86 1.248e-06 O═c1[nH]c2ccccc2c2ccc([N+] (═O)[O-])cc12

O═c1[nH]c2ccccc2c2ccc cc12

 87 1.248e-06 Cc1cccc(C2NC(═O)c3ccccc3 O2)c1

O═C1NC(c2ccccc2)Oc2 ccccc21

 88 1.248e-06 Oc1ccccclC1CC(c2ccccc2)N N1

c1ccc(C2CC(c3ccccc3)N N2)cc1

 89 1.248e-06 Cc1ccc(C2═NNC(═O)C3CCC CC32)cc1

O═C1NN═C(c2ccccc2)C 2CCCCC12

 90 1.248e-06 O═C1CCC(C2CCc3cc(O)ccc3 C2)C1

O═C1CCC(C2CCc3cccc c3C2)C1

 91 1.248e-06 OC1C(O)C2c3ccccc3C1c1ccc cc12

c1ccc2c(c1)C1CCC2c2c cccc21

 92 1.248e-06 O═C(Nc1ccc2nccnc2c1)c1ccc cc1

c1ccc2nccnc2c1

 93 1.248e-06 CC(═O)c1ccc(- c2ccc(C(N)═O)cn2)cc1

c1ccc(-c2ccccn2)cc1

 94 1.248e-06 O═c1oc2ccccc2c2ccc([N+](═ O)[O-])cc12

O═c1oc2ccccc2c2ccccc 12

 95 1.248e-06 O═C1c2ccccc2- c2ccc([N+](═O)[O-])cc21

O═C1c2ccccc2- c2ccccc21

 96 1.248e-06 O═C(Nc1n[nH]c2cc(F)ccc12)c 1ccoc1

c1ccc2[nH]ncc2c1

 97 1.248e-06 [O]S([O])([O])c1ccc2c(c1)oc1c cccc12

c1ccc2occc2c1

 98 1.248e-06 COC1Nc2ccccc2- c2ccccc2C1═O

O═C1CNc2ccccc2- c2ccccc21

 99 1.248e-06 O═C(Nc1ccccn1)c1ccc2c(c1) CCO2

c1ccc2c(c1)CCO2

100 1.248e-06 Cc1cc(═O)[nH]c(/C═C/c2cc(F) ccc2F)n1

O═c1ccnc(C═Cc2ccccc2) [nH]1

101 1.467e-06 Cc1cccc(- c2noc(C3CCCCN3)n2)c1

c1ccc(- c2noc(C3CCCCN3)n2)c c1

102 1.467e-06 Fc1ccc(- c2nnc3n2CCCCC3)cc1

c1ccc(- c2nnc3n2CCCCC3)cc1

103 1.467e-06 Cc1ccc(-c2nc(- c3nonc3N)no2)cc1

c1ccc(-c2nc(- c3cnon3)no2)cc1

104 1.467e-06 Nc1cccc(-c2nc(- c3ccccc3)no2)c1

c1ccc(-c2noc(- c3ccccc3)n2)cc1

105 1.467e-06 OC1CC(c2ccc(O)cc2)Oc2ccc cc21

c1ccc(C2CCc3ccccc3O2) cc1

106 1.467e-06 [O]c1ccccc1C(═O)n1ccc2cccc c21

O═C(c1ccccc1)n1ccc2cc ccc21

107 1.467e-06 Nc1ccc(C2N═C(c3ccccc3)[N] N2)cc1

c1ccc(C2═NC(c3ccccc3) N[N]2)cc1

108 1.467e-06 Cc1cc2nc(- c3ccc(N)cc3)[nH]c2cc1N

c1ccc(- c2nc3ccccc3[nH]2)cc1

109 1.467e-06 [O]C(═O)C1c2ccccc2- c2ccccc21

c1ccc2c(c1)Cc1ccccc1-2

110 1.467e-06 O═C1Cc2ccccc2C(c2ccccc2) N1

O═C1Cc2ccccc2C(c2ccc cc2)N1

111 1.467e-06 [NH]c1[nH]c([N]c2ccc(F)cc2)c 2ccccc12

c1ccc2c[nH]cc2c1

112 1.467e-06 O═C(c1ccccc1)c1c[nH]c2cccc c2c1═O

O═c1cc[nH]c2ccccc12

113 1.467e-06 Oc1cccc(-c2nc(- c3cccnc3)no2)c1

c1ccc(-c2nc(- c3cccnc3)no2)cc1

114 1.467e-06 Oc1cccc(-c2nc(- c3ccncc3)no2)c1

c1ccc(-c2nc(- c3ccncc3)no2)cc1

115 1.467e-06 Oc1ccc(-c2cccc(- n3cnnc3)c2)cc1

c1ccc(-n2cnnc2)cc1

116 1.467e-06 CC(NN)c1ccc2c(c1)C═C1C═ CC═CC12

C1═CC2═CC═CC2C═C1

117 1.467e-06 Cc1ccc(Nc2nc3[nH]ncc3c(═O) [nH]2)cc1

O═c1[nH]cnc2[nH]ncc12

118 1.467e-06 OC1CCCN(c2ncnc3cccc(F)c2 3)C1

c1ccc2c(N3CCCCC3)nc nc2c1

119 1.467e-06 Cc1ccc(- c2nc(C3CCOC3)no2)cc1F

c1ccc(- c2nc(C3CCOC3)no2)cc1

120 1.467e-06 O═C(c1cc[nH]c(═O)c1)N1CC C2CCCCC21

C1CCC2NCCC2C1

121 1.467e-06 Cc1ccc(- c2cc(═O)[nH]c(C3CC3)n2)cc1

O═c1cc (c2ccccc2)nc[nH]1

122 1.467e-06 CN/C(═N\c1ccc(C2═NNC(═O) CC2C)cc1)NC#N

O═C1CCC(c2ccccc2)═N N1

123 1.725e-06 Fc1cccc(-c2nnc(- c3cccnc3)o2)c1

c1ccc(-c2nnc(- c3cccnc3)o2)cc1

124 1.725e-06 OC1c2ccccc2Cc2ccccc21

c1ccc2c(c1)Cc1ccccc1C 2

125 1.725e-06 NC(═O)c1ccc2nc (c3ccccc3)oc2c1

c1ccc(- c2nc3ccccc3o2)cc1

126 1.725e-06 [O]C(═O)c1cccc (c2cccc(═O)[nH]2)c1F

O═c1cccc(- c2ccccc2)[nH]1

127 1.725e-06 Nc1nc2ccccc2n1/N═C/c1cccc c1

C(═Nn1cnc2ccccc21)c1c cccc1

128 1.725e-06 Oc1ccc(/N═C/c2ccc3c(c2)OC O3)cc1

C(═Nc1ccccc1)c1ccc2c (c1)OCO2

129 1.725e-06 Oc1ccc(-c2n[nH]c(- c3ccncc3)n2)cc1

c1ccc(-c2n[nH]c(- c3ccncc3)n2)cc1

130 1.725e-06 O═C(Nc1nnc2ccccn12)C1CC CCC1

c1ccn2cnnc2c1

131 1.725e-06 Nc1nccc(-c2cccc(- c3cc[nH]n3)c2)n1

c1ccc(-c2ccncn2)cc1

132 1.725e-06 O═C(c1nccc2ccccc12)N1CC (CF)C1

c1ccc2cnccc2c1

133 1.725e-06 CC(═O)Nc1cc2c(cc10)═C1C═ CC═CC1C═2

C1═CC2═c3ccccc3═CC 2C═C1

134 1.725e-06 CC1CN(C(═O)C(C)C)CC(c2c cccc2)O1

c1ccc(C2CNCCO2)cc1

135 2.028e-06 Fc1ccc(-c2nnc(- c3ccncc3)o2)cc1

c1ccc(-c2nnc(- c3ccncc3)o2)cc1

136 2.028e-06 [O]C(═O)c1nc(C2═Cc3ccccc3 OC2)no1

C1═Cc2ccccc2OC1

137 2.028e-06 O═C1C2C3CCC(C3)C2C(═O) C2C3CCC(C3)C21

O═C1C2C3CCC(C3)C2 C(═O)C2C3CCC(C3)C1 2

TABLE 13 Small molecule modulators targeting FNIP2 Exemplar Dissociation No Constants Exemplar SMILES Exemplar Image Scaffold SMILES Scaffold Image  1 4.019e-07 CCSC(C(C(═O)N(C)C(C(C)C)C(═O)O CC(NC(═O)c1cnc2ccccc2n1)C(═O)N C(C)C(═O)[N]C)N(C)C(═O)C(C)NC(═ O)C(COC[O])NC(═O)c1cnc2ccccc2n1) [S](C)CC1CC11

c1ccc2nccnc2c1

 2 5.555e-07 O═C(O)c1ccc2cc (c3ccc(O)c(C45CC6CC(CC(C6)C4)C5) c3)ccc2c1

c1ccc(- c2cccc(C34CC5CC (CC(C5)C3)C4)c2)cc1

 3 5.555e-07 Cc1ccc(C(═O)Nc2ccc(S([O])([O])O)c3 cc(S([O])([O])O)cc(S([O])([O])O)c23)c c1NC(═O)c1cccc(NC(═O)Nc2cccc(C (═O)Nc3cc(C(═O)Nc4ccc(S([O])([O])O) c5cc(S([O])([O])O)cc(S([O])([O])O)c4 5)ccc3C)c2)c1

c1ccc2ccccc2c1

 4 7.679e-07 COC(C(C)N(C)C(═O)c1ccccc1)[C@] (C)(OC)n1c2ccccc2c2c3c(c4c(c21)[N] C1CCCCC14)C(═O)NC3

O═C1NCc2ccccc21

 5 9.027e-07 CN1CC(/C(O)═N/[C@]2(C)O[C@@]3 (O)C4CCCN4C(═O)C(Cc4ccccc4)N3 C2═O)C═C2C1Cc1c[nH]c3cccc2c13

c1ccc2[nH]ccc2c1

 6 9.027e-07 O═C(NC1CCN(CCCCC2(C(═O)NCC (F)(F)F)c3ccccc3- c3ccccc32)CC1)c1ccccc1- c1ccc(C(F)(F)F)cc1

c1ccc2c(c1)- c1ccccc1C2CCCCN1 CCCCC1

 7 1.061e-06 CC(C)NC(═O)C1CCC(n2c(NC(═O)c3 ccc(F)cc3)nc3ccc(CN4CCC(C(C)(C) O)CC4)cc32)CC1

c1ccc2[nH]cnc2c1

 8 1.061e-06 COc1ccc2c(OC3CC4C(═O)N[C@]5 (C(═O)O)CC5/C═C/CCCCCC(NC(═O) OC5CCCC5)C(═O)N4C3)cc(C3CSC (NC(C)C)[N]3)nc2c1

O═C1NCCC═CCCC CCCC(═O)N2CCCC 12

 9 1.248e-06 CN(C(Cc1ccc(OS([O])([O])c2cccc3c2 C2CN2C3)cc1)C(═O)N1CCN(c2ccccc 2)CC1)S(═O)(═O)c1cccc2c1C1CN1C 2

C1═NCc2ccccc21

10 1.248e-06 COCOC1CCC(O)COC/C1═C\C═C\C (C)[C@]([O])(OC1CCC(OC2CC(OC) [C@]3(CN3)C(C)O2)C(C)O1)C(C)C(C) C1CC1C10[C@@]12C═CC(C)C(C (C)C)O2

C1═CC2(OCC1)OC2 C1CC1CCCOC1CC C(OC2CCCCO2)CO 1

11 1.248e-06 O═C(O)c1cc(O)c2c(O)c3c(OC4OC(C O)C(O)C(O)C4O)cccc3c(- c3c4cc(C(═O)O)cc(O)c4c(O)c4c(OC5 OC(CO)C(O)C(O)C5O)cccc34)c2c1

c1ccc2c(- c3c4ccccc4cc4c(OC 5CCCCO5)cccc34)c 3cccc(OC4CCCCO4) c3cc2c1

12 1.725e-06 CN(C)C/C═C/C(═O)Nc1cccc(C(═O)N c2cccc(Nc3ncc(Cl)c(C4CNc5ccccc54) n3)c2)c1

O═C(Nc1cccc(Nc2nc cc(C3CNc4ccccc43) n2)c1)c1ccccc1

13 1.725e-06 CC(C)C1NC(═O)C(NC(═O)C2C(N)C (═O)C(C)C3═C2N═C2C(O3)C(C)═CC ═C2C(═O)NC2C(C)OC(═O)C(C(C)C) N(C)C(═O)CN(C)C(═O)C3CCCN3C(═ O)C(C(C)C)NC2═O)C(C)OC(═O)C(C (C)C)N(C)C(═O)CN(C)C(═O)C2CCCN 2C1═O

O═C1CC2═C(N═C3 C(C(═O)NC4COC(═ O)CNC(═O)CNC(═O) C5CCCN5C(═O)CN C4═O)═CC═CC3O2) C(C(═O)NC2COC(═ O)CNC(═O)CNC(═O) C3CCCN3C(═O)CN C2═O)C1

14 2.028e-06 CC1CCC(OC2CC(O)C(OC3CC(O)C (OC4CC(O)C(O)C(C)O4)C(C)O3)C(C) O2)CC1CCC1CCC(O)[C@]2(C)C[C @]120

C1CCC2CC2C1

15 2.028e-06 CC(CCN(C)Cc1ccccn1)n1cc(C2═C(c 3cn(C)c4ccccc34)C(═O)NC2═O)c2cc ccc21

O═C1C═C(c2c[nH]c3 ccccc23)C(═O)N1

16 2.384e-06 COc1cc2c(cc1OC)CN(CCc1ccc(N3[N] N═C(c4cc(OC)c(OC)cc4NC(═O)C4C C(═O)c5ccccc5O4)[N]3)cc1)CC2

c1ccc2c(c1)CCNC2

17 2.384e-06 CCN1CCN(c2ccc3cc(- c4cn5cc(C)nc(C)c5n4)oc(═O)c3c2)C C1C

c1cn2ccnc2cn1

18 2.384e-06 Cc1csc(-c2nnc(Nc3ccc(Oc4ncccc4- c4ccnc(N)n4)cc3)c3ccccc23)c1

c1ccc2cnncc2c1

19 2.384e-06 OC(c1ccc(- c2ccc(CN3CCN(Cc4ccncc4)CC3)cc2) c(F)c1)(C(F)(F)F)C(F)(F)F

c1ccc(-c2ccccc2)cc1

20 2.384e-06 O═C(/C═C\N1CN═C(c2cc(C(F)(F)F)c c(C(F)(F)F)c2)[N]1)NNc1ccccn1

c1ccc(C2═NCN[N]2) cc1

21 2.803e-06 CC1CC(OC(═O)C(O)C(/N═C(/O)C2═ CC═CCC2)C2═CCCCC2)[C@@]12C C2(O)COC(═O)c1ccccc1

C1CC2(C1)CC2

22 2.803e-06 CC(C)CC(C([O])[N]OC(C)[C@]1([O]) C(C(CC(C)C)C2(OC(CCc3ccc(N4CC OCC4)cc3)C(═O)[N][C@@]3(CC(C)C) OC3C(C)[O])OO2)N1C)N(C)C(C)[O]

c1ccc(N2CCOCC2)c c1

23 2.803e-06 Cc1ccc2cc(-c3c(C)ccc(C(═O)c4c(- c5ccccc5)n(C)n(- c5ccccc5)c4═O)c3N)ccc2n1

c1ccc2ncccc2c1

24 2.803e-06 C/C═C\[N]C[C@@11(C2CCN(Cc3cnc c(C)c3)CC2)CCCc2cc(Cl)ccc21

c1ccc2c(c1)CCCC2

25 2.803e-06 CCOC(CO)Oc1coc2cc(O)cc(O)c2c1═ O

O═c1ccoc2ccccc12

26 2.803e-06 CCc1cc(Nc2nccc(-c3c(- c4ccc(OC)c(C(═O)Nc5c(F)cccc5F)c4) nc4ccccn34)n2)c(OC)cc1N1CCC(N2 CCN(S(C)(═O)═O)CC2)CC1

c1ccn2ccnc2c1

27 3.295e-06 CN(C)C(═O)C(CCN1CCC(O)(c2ccc (Cl)cc2)CC1)(c1ccccc1)c1ccccc1

c1ccc(Cc2ccccc2)cc 1

28 3.295e-06 CC1CC2C[C@](C)(CCC(O)C(C)═O)C CC2[C@]2(C)C3C[C@]3([O])C═C12

C1CCC2CCCCC2C1

29 3.295e-06 [O]C(OC1COc2cc(O)cc(O)c2C1═O)C (O)CO

O═C1CCOc2ccccc21

30 3.295e-06 CC(C)(/C═C(C#N)C(═O)N1CCCC(n2 nc(- c3ccc(Oc4ccccc4)cc3F)c3c(N)ncnc32) C1)N1CCN(C2COC2)CC1

c1ncc2cnn(C3CCCN C3)c2n1

31 3.295e-06 COC1/C═C/O[C@@]2(C)Oc3c(C)c(O) c4c(O)c(c(C[N][N]CN5CCCCC5)c(O) c4c3C2═O)NC(═O)/C(C)═C\C═C\C(C) C(O)C(C)C(O)C(C)C(OC(C)═O)C1c

O═C1C═CC═CCCC CCCCCC═COC2Oc3 ccc4cc(ccc4c3C2═O) N1

32 3.295e-06 COc1cc2c(cc1Oc1c3c(cc4c1C(CC1C CCCC1)N(C)CC4)OCO3)C(CC1CC[ C@]3(OC)CC13)N(C)CC2

c1ccc2c(c1)OCO2

33 3.874e-06 CCC(CCC(C)C1CCC2C3CC═C4CC( OC5OC(CO)C(O)C(O)C5O)CC[C@]4 (C)C3CC[C@]12C)C(C)C

C1═C2CCCCC2C2C CC3CCCC3C2C1

34 3.874e-06 Cc1ccc(NC(═O)c2ccc(CN3CCN(C)C C3)cc2)cc1Nc1nccc(-c2cccnc2)n1 Cc1ccc(C(═O)Nc2ccc(CN3CCN(C)C

c1cncc(-c2ccncn2)c1

35 3.874e-06 Ccc1ccc(C═O)Nc2ccc(CN3CCN(C)C C3)c(C(F)(F)F)c2cc1C#Cc1cnc2cccn n12

c1cnn2ccnc2c1

36 3.874e-06 O═C1CCc2c(Oc3ccc4c(c3)C3C(O4)C 3c3nc4ccc(C(F)(F)F)cc4[nH]3)ccnc2 N1

O═C1CCc2c(Oc3ccc cc3)ccnc2N1

37 3.874e-06 CCOC(OCC[C@@]2(O)CCC3C(C( O)C[C@](C)(C(C)C4═CC(═O)OC4)[C @]3(C)O)[C@@]23CC3O)C(O)C(O) C1O

C1CCC2C(C1)CCCC 21CC1

38 3.874e-06 COc1ccc(C2CC(═O)c3c(O)cc(OC4O C(C)C(OCOC(CO)CO)C(O)C4O)cc3 O2)cc10

O═C1CC(c2ccccc2) Oc2cc(OC3CCCCO3) ccc21

39 3.874e-06 CC1OC(OC2C(OC3C[C@]4(C)C(CC( O)C5C([C@](C)(CCC═C(C)C)OC6O C(CO)C(O)C(O)C6O)CC[C@]54C)[C @@]4(C)CCC(O)C(C)(C)C34)OC(CO) C(O)C2O)C(O)C(O)C1O

C1CCC2C(C1)CCC1 CCCCC12

40 4.554e-06 CC(C)NC(═O)COc1cccc(C2═NC(Nc3 ccc4n[nH]cc4c3)c3ccccc3[N]2)c1

c1ccc2n[nH]cc2c1

41 4.554e-06 CC[C@]1(C[C@]23CC[C@]4(C)C(C5 ═CC(═O)OC5)CC[C@]4(O)C2C3)CC C(OC2CC(O)C(OC3CC(O)C(OC4CC (O)C(O)C(C)O4)C(C)O3)C(C)O2)C1

C1CCC2CCCC2C1

42 4.554e-06 Cc1ccc(NC(═O)C2(c3ccc4c(c3)OC(F) (F)O4)CC2)nc1-c1cccc(C(═O)O)c1 CCCc1cc(C)[nH]c(═O)c1CNC(═O)c1 c

c1cc2c(cc1C1CC1)O CO2

43 4.554e-06 c(- c2ccc(N3CCN(C(C)C)CC3)nc2)cc2c1 cnn2C(C)C

c1ccc2[nH]ncc2c1

44 4.554e-06 CC1(C)CCC(CN2CCN(c3ccc(C(═O)N S(═O)(═O)c4ccc(NCC5CCOCC5)c([N+] (═O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c 3)CC2)═C(c2ccc(CI)cc2)C1

c1cnc2[nH]ccc2c1

45 5.354e-06 COC1CN(C2CC(OC3C[C@](O)(C(═ O)CO)Cc4c(O)c5c(c(O)c43)C(═O)c3c (OC)cccc3C5═O)OC(C)C2O)CCO1

O═C1c2ccccc2C(═O) c2cc3c(cc21)CCCC3 OC1CCCCO1

46 5.354e-06 Cc1cc(Nc2ncnc3ccc(NC4═NC(C)(C) CO4)cc23)ccc1Oc1ccn2ncnc2c1

c1ccc2c(Nc3ccc(Oc4 ccn5ncnc5c4)cc3)nc nc2c1

47 5.354e-06 CC1CCC2C(C═C(C)C3CC(═O)CC[C @@]23C)C1CCC(OC(C)═O)C(C)═O

C1═CC2CCCCC2CC 1

48 5.354e-06 Cc1[nH]c(/C═C2\C(═O)Nc3ccc(S(═O) (═O)N(C)c4cccc(CI)c4)cc32)c(C)c1C (═O)N1CCN(C)CC1

C═C1C(═O)Nc2cccc c21

49 5.354e-06 CC(═O)[C@]12O[C@](C)(c3ccccc3) OC1CC1C3CCC4CC(═O)CC[C@@]4 (C)C3CC[C@@]12C

O═C1CCC2C(CCC3 C2CCC2C3CC3OCO C32)C1

50 5.354e-06 CC(CCCC(O)(C(F)(F)F)C(F)(F)F)C1 CCC2/C(═C/C═C3/CC(O)CC(O)C3═ C)CCC[C@]12C

C═C1CCCC2CCCC1 2

51 6.295e-06 COC1CC(O)C(OC2OC(COS([O])([O]) O)C(OC3OC(C(═O)O)C(OC4OC(CO S([O])([O])O)C(O)C(O)C4NS([O])([O]) O)C(O)C3O)C(OS([O])([O])O)C2NS ([O])([O])O)C(C(═O)O)O1

C1CCC(OC2CCCOC 2)OC1

52 6.295e-06 Cn1cc( c2cc3c(N4CCN(c5ncc([C@@](C)(N)c 6ccc(F)cc6)cn5)CC4)ncnn3c2)cn1

c1ccc(Cc2cnc(N3CC N(c4ncnn5cc(- c6cn[nH]c6)cc45)CC 3)nc2)cc1

53 6.295e-06 CCOC(═O)O[C@]([O])(COC(═O)CC) C1C[C@@]12C1CC(O)C3C(CCC4C C(═O)CC[C@]43C)C12

C1CCC2C(C1)C21C C1

54 6.295e-06 COc1cnc(N2CN═C(C)[N]2)c2c1c(C(═ O)C(═O)N1CCN(C(═O)c3ccccc3)CC1 )cn2COP(═O)(O)O

O═C(C(═O)N1CCN (C(═O)c2ccccc2)CC1) c1c[nH]c2c(N3CN═C [N]3)nccc12

55 6.295e-06 CCN1CCC(N(C)C2N═C(C3CCCCC3) [N]c3cc(OCCCN4CCCC4)c(OC)cc32) CC1

C1═NCc2ccccc2[N]1

56 6.295e-06 O═C(OC(C(F)(F)F)C(F)(F)F)N1CCC (C(O)(c2ccc3c(c2)OCO3)c2ccc3c(c2) OCO3)CC1

c1cc2c(cc1C(c1ccc3 c(c1)OCO3)C1CCNC C1)OCO2

57 6.295e-06 CCC(CC(C)F)C1C(C(C)N2[N]C(c3cc c(OC(C)C)c(F)c3)c3c(N)ncnc32)Oc2c cc(F)cc2C1═O

c1ncc2c(n1)N[N]C2

58 7.400e-06 CC(C)n1c(═O)n(C)c2cnc3cc(F)c(- c4ccc(OCCCN5CCCCC5)nc4)cc3c21

O═c1[nH]c2cnc3ccc(- (c4cccnc4)cc3c2[nH]1

59 7.400e-06 CCC1OC(═O)C(C)C(═O)C(C)C(OC2 OC(C)CC(N(C)C)C2O)[C@](C)(OC)C C(C)C(═O)C(C)C2N(CCCCn3cnc(- c4cccnc4)c3)C(═O)O[C@]12C

O═C1CCCCCC(═O) CC2NC(═O)OC2CO C(═O)C1

60 7.400e-06 C[C@]12CCC(═O)CC1CCC1C3C[C @@]4(C)[C@@](O)([C@@]3(C)CC (O)[C@]12F)[C@]4([O])CO

O═C1CCC2C(CCC3 CCCCC32)C1

61 8.701e-06 O═C(Nc1cc(-c2ccccc2)nn1- c1ccccc1F)c1ccc([N+](═O)[O-])cc1

c1ccc(- c2cc[nH]n2)cc1

62 8.701e-06 O═C(/C═C/N1CC2CC1CN2c1ccccnl) c1ccccc1O

C1NC2CNC1C2

63 8.701e-06 COc1ccc(N(C(═O)Oc2c(C)cccc2C)c2 ccnc(Nc3ccc(N4CCN(C)CC4)cc3)n2) c(OC)c1

c1ccc(N2CCNCC2)c c1

64 8.701e-06 NC(═O)c1ccc(N(C(N)═O)c2c(F)cccc2 F)nc1-c1ccc(F)cc1F

c1ccc(-c2ccccn2)cc1

65 8.701e-06 COc1ccc(C2[N]C(c3ccc(CI)cc3)C(c3c cc(CI)cc3)N2C(═O)N2CCNC(═O)C2)c (OC(C)C)c1

c1ccc(C2[N]C(c3cccc c3)C(c3ccccc3)N2)cc 1

66 1.023e-05 CC1CN(c2ccc(NC(═O)c3cccc(- c4ccc(OC(F)(F)F)cc4)c3C)cn2)CC(C) O1

c1ccc(N2CCOCC2)n c1

67 1.203e-05 COc1cc2nccc(Oc3ccc(NC(═O)c4cn(C (C)C)c(═O)n (c5ccc(F)cc5)c4═O)cc3F)c2cc1OC

c1ccc(Oc2ccnc3cccc c23)cc1

TABLE 14 Antibody modulators targeting FNIP1 and/or FNIP2 Number Catalogue ID Vendor Target 1 ABIN5066851 antibodies-online.com FNIP1 2 ABIN5066854 antibodies-online.com FNIP1 3 ABIN5066849 antibodies-online.com FNIP1 4 ABIN5066844 antibodies-online.com FNIP1 5 ABIN5066843 antibodies-online.com FNIP1 6 ABIN5066846 antibodies-online.com FNIP1 7 ABIN5066845 antibodies-online.com FNIP1 8 ABIN5066847 antibodies-online.com FNIP1 9 ABIN5066842 antibodies-online.com FNIP1 10 ABIN5066841 antibodies-online.com FNIP1 11 ABIN5066840 antibodies-online.com FNIP1 12 ABIN5066853 antibodies-online.com FNIP1 13 ABIN5066852 antibodies-online.com FNIP1 14 ABIN5066848 antibodies-online.com FNIP1 15 ABIN5066841 antibodies-online.com FNIP1 16 ABIN5066840 antibodies-online.com FNIP1 17 ABIN5066853 antibodies-online.com FNIP1 18 ABIN540656 antibodies-online.com FNIP1 19 ABIN238670 antibodies-online.com FNIP1 20 ABIN6930398 antibodies-online.com FNIP1 21 ABIN6930396 antibodies-online.com FNIP1 22 ABIN6930397 antibodies-online.com FNIP1 23 ABIN6930395 antibodies-online.com FNIP1 24 ABIN6930394 antibodies-online.com FNIP1 25 ABIN6930393 antibodies-online.com FNIP1 26 ABIN5514775 antibodies-online.com FNIP1 27 ABIN5697699 antibodies-online.com FNIP1 28 ABIN6081543 antibodies-online.com FNIP1 29 ABIN6081545 antibodies-online.com FNIP1 30 ABIN5066850 antibodies-online.com FNIP1 31 ABIN6081547 antibodies-online.com FNIP1 32 ABIN6081549 antibodies-online.com FNIP1 33 ABIN1042594 antibodies-online.com FNIP1 34 ABIN2268375 antibodies-online.com FNIP1 35 ABIN2268376 antibodies-online.com FNIP1 36 ABIN2268377 antibodies-online.com FNIP1 37 ABIN2268378 antibodies-online.com FNIP1 38 ABIN2268380 antibodies-online.com FNIP1 39 ABIN2268379 antibodies-online.com FNIP1 40 ABIN341163 antibodies-online.com FNIP1 41 abx917051 abbexa.com FNIP1 42 abx917052 abbexa.com FNIP1 43 abx313611 abbexa.com FNIP1 44 abx233179 abbexa.com FNIP1 45 abx432703 abbexa.com FNIP1 46 abx448561 abbexa.com FNIP1 47 abx447818 abbexa.com FNIP1 48 abx447819 abbexa.com FNIP1 49 abx447810 abbexa.com FNIP1 50 abx447811 abbexa.com FNIP1 51 abx447813 abbexa.com FNIP1 52 abx313614 abbexa.com FNIP1 53 abx447820 abbexa.com FNIP1 54 abx313613 abbexa.com FNIP1 55 abx447821 abbexa.com FNIP1 56 abx313612 abbexa.com FNIP1 57 abx447822 abbexa.com FNIP1 58 abx447824 abbexa.com FNIP1 59 abx447825 abbexa.com FNIP1 60 abx964266 abbexa.com FNIP1 61 abx387393 abbexa.com FNIP1 62 abx981023 abbexa.com FNIP1 63 abx524104 abbexa.com FNIP1 64 LS-C82348 LifeSpan BioSciences, Inc. FNIP1 65 LS-C61738 LifeSpan BioSciences, Inc. FNIP1 66 LS-C665632 LifeSpan BioSciences, Inc. FNIP1 67 LS-C677965 LifeSpan BioSciences, Inc. FNIP1 68 LS-C677967 LifeSpan BioSciences, Inc. FNIP1 69 LS-C677969 LifeSpan BioSciences, Inc. FNIP1 70 LS-C677968 LifeSpan BioSciences, Inc. FNIP1 71 LS-C763277 LifeSpan BioSciences, Inc. FNIP1 72 LS-C773233 LifeSpan BioSciences, Inc. FNIP1 73 LS-C773235 LifeSpan BioSciences, Inc. FNIP1 74 LS-C773234 LifeSpan BioSciences, Inc. FNIP1 75 LS-C773237 LifeSpan BioSciences, Inc. FNIP1 76 LS-C773239 LifeSpan BioSciences, Inc. FNIP1 77 LS-C773240 LifeSpan BioSciences, Inc. FNIP1 78 LS-C773238 LifeSpan BioSciences, Inc. FNIP1 79 LS-C773236 LifeSpan BioSciences, Inc. FNIP1 80 LS-C750519 LifeSpan BioSciences, Inc. FNIP1 81 LS-C775781 LifeSpan BioSciences, Inc. FNIP1 82 LS-C775783 LifeSpan BioSciences, Inc. FNIP1 83 LS-C775784 LifeSpan BioSciences, Inc. FNIP1 84 LS-C775786 LifeSpan BioSciences, Inc. FNIP1 85 LS-C775787 LifeSpan BioSciences, Inc. FNIP1 86 LS-C775788 LifeSpan BioSciences, Inc. FNIP1 87 LS-C775785 LifeSpan BioSciences, Inc. FNIP1 88 LS-C775789 LifeSpan BioSciences, Inc. FNIP1 89 LS-C775780 LifeSpan BioSciences, Inc. FNIP1 90 PA5-77778 Invitrogen Antibodies FNIP1 91 PA5-71423 Invitrogen Antibodies FNIP1 92 PA5-64359 Invitrogen Antibodies FNIP1 93 PA5-64453 Invitrogen Antibodies FNIP1 94 19847-1-AP Proteintech Group FNIP1 95 28380-1-AP Proteintech Group FNIP1 96 NB100-93396 Novus Biologicals FNIP1 97 NBP2-49544 Novus Biologicals FNIP1 98 NBP2-49647 Novus Biologicals FNIP1 99 NBP2-87460 Novus Biologicals FNIP1 100 GTX82555 GeneTex FNIP1 101 GTX88818 GeneTex FNIP1 102 110739 NovoPro Biosciences Inc. FNIP1 103 orb156915 Biorbyt FNIP1 104 orb45245 Biorbyt FNIP1 105 orb448990 Biorbyt FNIP1 106 orb470827 Biorbyt FNIP1 107 orb19734 Biorbyt FNIP1 108 orb45246 Biorbyt FNIP1 109 orb45247 Biorbyt FNIP1 110 orb45248 Biorbyt FNIP1 111 orb462210 Biorbyt FNIP1 112 orb486703 Biorbyt FNIP1 113 orb543061 Biorbyt FNIP1 114 orb55594 Biorbyt FNIP1 115 orb55595 Biorbyt FNIP1 116 orb55596 Biorbyt FNIP1 117 orb55597 Biorbyt FNIP1 118 orb55598 Biorbyt FNIP1 119 orb55599 Biorbyt FNIP1 120 orb55600 Biorbyt FNIP1 121 orb55601 Biorbyt FNIP1 122 orb55602 Biorbyt FNIP1 123 orb55603 Biorbyt FNIP1 124 orb55604 Biorbyt FNIP1 125 orb55605 Biorbyt FNIP1 126 orb55606 Biorbyt FNIP1 127 orb55607 Biorbyt FNIP1 128 orb55608 Biorbyt FNIP1 129 orb55609 Biorbyt FNIP1 130 orb55610 Biorbyt FNIP1 131 orb55611 Biorbyt FNIP1 132 PAB12367 Abnova Corporation FNIP1 133 PAB7550 Abnova Corporation FNIP1 134 H00096459-K Abnova Corporation FNIP1 135 HPA068099 Atlas Antibodies FNIP1 136 HPA071213 Atlas Antibodies FNIP1 137 CBMAB-F0933-CQ Creative Biolabs FNIP1 138 MOR-1329 Creative Biolabs FNIP1 139 DCABH-15436 Creative Diagnostics FNIP1 140 CPBT-33934RH Creative Diagnostics FNIP1 141 DPAB-DC3873 Creative Diagnostics FNIP1 142 STJ117747 St. John's Laboratory FNIP1 143 STJ71460 St. John's Laboratory FNIP1 144 ab13469 RabMAbs FNIP1 145 MBS2529883 MyBioSource FNIP1 146 MBS421591 MyBioSource FNIP1 147 AP03045SU-N Acris Antibodies GmbH FNIP1 148 OAEB00546 Aviva Systems Biology FNIP1 149 EB08326 Everest Biotech FNIP1 150 AF2703a Abgent FNIP1 151 APG00517G Leading Biology FNIP1 152 46-659 ProSci FNIP1 153 abx917053 abbexa.com FNIP2 154 abx917054 abbexa.com FNIP2 155 abx961571 abbexa.com FNIP2 156 abx524105 abbexa.com FNIP2 157 abx983570 abbexa.com FNIP2 158 abx524106 abbexa.com FNIP2 159 ABIN952349 antibodies-online.com FNIP2 160 ABIN615692 antibodies-online.com FNIP2 161 ABIN3092610 antibodies-online.com FNIP2 162 ABIN3135978 antibodies-online.com FNIP2 163 ABIN6656599 antibodies-online.com FNIP2 164 ABIN5578275 antibodies-online.com FNIP2 165 ABIN499853 antibodies-online.com FNIP2 166 ABIN5578273 antibodies-online.com FNIP2 167 ABIN1734392 antibodies-online.com FNIP2 168 ABIN1030400 antibodies-online.com FNIP2 169 ABIN6656600 antibodies-online.com FNIP2 170 ABIN499855 antibodies-online.com FNIP2 171 ABIN1734393 antibodies-online.com FNIP2 172 ABIN1031382 antibodies-online.com FNIP2 173 ABIN2721270 antibodies-online.com FNIP2 174 ABIN1382786 antibodies-online.com FNIP2 175 ABIN1382790 antibodies-online.com FNIP2 176 ABIN2861625 antibodies-online.com FNIP2 177 ABIN2861626 antibodies-online.com FNIP2 178 ABIN1812731 antibodies-online.com FNIP2 179 ABIN2268382 antibodies-online.com FNIP2 180 ABIN2268387 antibodies-online.com FNIP2 181 ABIN2268389 antibodies-online.com FNIP2 182 ABIN2268391 antibodies-online.com FNIP2 183 ABIN2268390 antibodies-online.com FNIP2 184 ABIN2268388 antibodies-online.com FNIP2 185 ABIN2268392 antibodies-online.com FNIP2 186 ABIN2602382 antibodies-online.com FNIP2 187 ABIN1896209 antibodies-online.com FNIP2 188 ABIN1896208 antibodies-online.com FNIP2 189 ABIN1896210 antibodies-online.com FNIP2 190 ABIN1896211 antibodies-online.com FNIP2 191 ABIN1896213 antibodies-online.com FNIP2 192 ABIN1896212 antibodies-online.com FNIP2 193 LS-B4600 LifeSpan BioSciences, Inc. FNIP2 194 LS-B4598 LifeSpan BioSciences, Inc. FNIP2 195 LS-C166200 LifeSpan BioSciences, Inc. FNIP2 196 LS-C235336 LifeSpan BioSciences, Inc. FNIP2 197 LS-C235337 LifeSpan BioSciences, Inc. FNIP2 198 LS-C235338 LifeSpan BioSciences, Inc. FNIP2 199 LS-C235340 LifeSpan BioSciences, Inc. FNIP2 200 LS-C235339 LifeSpan BioSciences, Inc. FNIP2 201 LS-C321416 LifeSpan BioSciences, Inc. FNIP2 202 LS-C248999 LifeSpan BioSciences, Inc. FNIP2 203 PAB16703 Abnova Corporation FNIP2 204 PAB16707 Abnova Corporation FNIP2 205 PAB23979 Abnova Corporation FNIP2 206 H00057600-B01P Abnova Corporation FNIP2 207 orb28569 Biorbyt FNIP2 208 orb75149 Biorbyt FNIP2 209 orb89825 Biorbyt FNIP2 210 orb89827 Biorbyt FNIP2 211 orb75153 Biorbyt FNIP2 212 orb156916 Biorbyt FNIP2 213 orb28568 Biorbyt FNIP2 214 orb448991 Biorbyt FNIP2 215 orb470828 Biorbyt FNIP2 216 orb462211 Biorbyt FNIP2 217 orb486704 Biorbyt FNIP2 218 AP51694PU-N OriGene FNIP2 219 AP23472PU-N OriGene FNIP2 220 AP23474PU-N OriGene FNIP2 221 TA306730 OriGene FNIP2 222 TA306734 OriGene FNIP2 223 AP51694PU-N Acris Antibodies GmbH FNIP2 224 AP51694PU-N Acris Antibodies GmbH FNIP2 225 AP51694PU-N Acris Antibodies GmbH FNIP2 226 AP51694PU-N Acris Antibodies GmbH FNIP2 227 AP51694PU-N Acris Antibodies GmbH FNIP2 228 AP51694PU-N Acris Antibodies GmbH FNIP2 229 AP51694PU-N Acris Antibodies GmbH FNIP2 230 PA5-24150 Invitrogen Antibodies FNIP2 231 PA5-20690 Invitrogen Antibodies FNIP2 232 PA5-20691 Invitrogen Antibodies FNIP2 233 PA5-111162 Invitrogen Antibodies FNIP2 234 PA5-111693 Invitrogen Antibodies FNIP2 235 GTX85348 GeneTex FNIP2 236 GTX85347 GeneTex FNIP2 237 LS-B4598 LifeSpan BioSciences, Inc. FNIP2 238 LS-B4600 LifeSpan BioSciences, Inc. FNIP2 239 LS-C166200 LifeSpan BioSciences, Inc. FNIP2 240 LS-C235336 LifeSpan BioSciences, Inc. FNIP2 241 LS-C235337 LifeSpan BioSciences, Inc. FNIP2 242 LS-C235338 LifeSpan BioSciences, Inc. FNIP2 243 LS-C235339 LifeSpan BioSciences, Inc. FNIP2 244 LS-C235340 LifeSpan BioSciences, Inc. FNIP2 245 LS-C248999 LifeSpan BioSciences, Inc. FNIP2 246 LS-C321416 LifeSpan BioSciences, Inc. FNIP2 247 LS-C553194 LifeSpan BioSciences, Inc. FNIP2 248 LS-C572845 LifeSpan BioSciences, Inc. FNIP2 249 LS-C592499 LifeSpan BioSciences, Inc. FNIP2 250 LS-C612149 LifeSpan BioSciences, Inc. FNIP2 251 LS-C631799 LifeSpan BioSciences, Inc. FNIP2 252 OAAB00671 Aviva Systems Biology FNIP2 253 134796 NovoPro Bioscience Inc. FNIP2 254 NBP1-93724 Novus Biologicals FNIP2 255 NBP2-68936 Novus Biologicals FNIP2 256 600-401-BF8 Rockland Immunochemicals, FNIP2 Inc. 257 600-401-BF9 Rockland Immunochemicals, FNIP2 Inc. 258 APS10854 Abgent FNIP2 259 APS10858 Abgent FNIP2 260 ALS13522 Abgent FNIP2 261 5045 ProSci FNIP2 262 5057 ProSci FNIP2 263 57612 Cell Signaling Technology, FNIP2 Inc. 264 HPA042779 Atlas Antibodies FNIP2 265 HPA072420 Atlas Antibodies FNIP2 266 MBS9209931 MyBioSource FNIP2 267 MBS150131 MyBioSource FNIP2 268 35705 United States Biological FNIP2 269 035705-AP United States Biological FNIP2 270 035705-APC United States Biological FNIP2 271 035705-Biotin United States Biological FNIP2 272 035705-FITC United States Biological FNIP2 273 035705-ML405 United States Biological FNIP2 274 035705-ML490 United States Biological FNIP2 275 035705-ML550 United States Biological FNIP2 276 035705-ML650 United States Biological FNIP2 277 035705-ML750 United States Biological FNIP2 278 035705-PE United States Biological FNIP2 279 35705 United States Biological FNIP2 280 035705-HRP United States Biological FNIP2 281 119-14583 RayBiotech, Inc. FNIP2 282 119-14585 RayBiotech, Inc. FNIP2 283 bs-13194R Bioss FNIP2 284 bs-13194R-Biotin Bioss FNIP2 285 bs-13194R-HRP Bioss FNIP2 286 bs-13194R-A350 Bioss FNIP2 287 bs-13194R-A488 Bioss FNIP2 288 bs-13194R-A555 Bioss FNIP2 289 bs-13194R-A647 Bioss FNIP2 290 bs-13194R-Cy3 Bioss FNIP2 291 bs-13194R-Cy5 Bioss FNIP2 292 bs-13194R-Cy5.5 Bioss FNIP2 293 bs-13194R-Cy7 Bioss FNIP2 294 bs-13194R-FITC Bioss FNIP2 295 254026 Abbiotec FNIP2 296 254030 Abbiotec FNIP2 297 DPABH-10189 Creative Diagnostics FNIP2 298 DPABH-10209 Creative Diagnostics FNIP2 299 AMM04573G Leading Biology FNIP2 300 AMM04574G Leading Biology FNIP2 301 AMM04572G Leading Biology FNIP2 302 CBMAB-CP073-LY Creative Biolabs FNIP2 303 SAB3500007 Sigma-Aldrich FNIP2 304 SAB3500010 Sigma-Aldrich FNIP2 

What is claimed is:
 1. A compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary to an equal length portion of SEQ ID NO: 1 or SEQ ID NO: 11, wherein the thymine bases are optionally uracil bases, and wherein the oligonucleotide comprises at least one modified sugar, at least one modified internucleoside linkage, or at least one modified nucleobase.
 2. The compound of claim 1, wherein the modified oligonucleotide consists of at least 8, at least 9, at least 10, at least 11, or at least 12 consecutive nucleobases with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity to any one of the nucleobase sequences of SEQ ID NOs: 16-1494.
 3. The compound of claim 1, wherein the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 16, 19, 20, 22, 23, 26, 28, 30, 43, 49, 70, 148, 155, 191, 259, 287, 304, 349, 385, 477, 593, 787, 804, 865, 912, 975, 978, 1083, 1085, 1197, 1198, 1199, 1200, 1202, 1205, and
 1206. 4. The compound of claim 1, wherein the modified oligonucleotide comprises at least one 2′-O-methoxyethyl (2′-MOE) modified sugar.
 5. The compound of claim 1, wherein the modified oligonucleotide comprises at least one 2′-O-methyl (2′-OMe) modified sugar.
 6. The compound of claim 1, wherein the modified oligonucleotide comprises at least one bicyclic sugar.
 7. The compound of claim 6, wherein each bicyclic sugar comprises a chemical bridge between the 4′ and 2′ positions of the sugar, wherein each chemical bridge is independently selected from: 4′-CH(R)—O-2′ and 4′ (CH₂)₂—O-2′, wherein R is independently selected from H, C₁-C₁₂ alkyl, or a protecting group.
 8. The compound of claim 1, wherein the modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage.
 9. The compound of claim 8, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.
 10. The compound of claim 8, wherein the modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.
 11. The compound of claim 1, wherein the modified oligonucleotide comprises at least one 5-methylcytosine modified nucleobase.
 12. The compound of claim 1, wherein the modified oligonucleotide comprises a gapped sequence, which gapped sequence comprises: a central sequence of linked deoxynucleosides; and wing sequences flanking both the 5′ and the 3′ ends of the central sequence, wherein at least one nucleoside of the wing sequences comprises a modified sugar.
 13. The compound of claim 12, wherein the central sequence is chosen to consist of 6, 7, 8, 9, 10, 11 or 12 linked nucleosides and the wing sequences are each independently chosen to consist of 3, 4, 5, or 6 linked nucleosides.
 14. The compound of claim 12, wherein the central sequence consists of 10 linked nucleosides and the wing sequences each consist of 4 linked nucleosides.
 15. The compound of claim 12, wherein the wing sequences comprise at least one nucleoside consisting of a 2′-O-methoxyethyl modified sugar.
 16. A composition comprising the compound of claim 1, or salt thereof, and at least one of a pharmaceutically acceptable carrier or diluent.
 17. A method comprising administering the compound of claim 1, or a pharmaceutical composition comprising the compound of claim 1, to a cell, animal, or human.
 18. A method to treat, prevent or ameliorate a TDP-43 proteinopathy, neurodegenerative or neuromuscular disease in a subject, comprising administering the compound of claim 1, or a pharmaceutical composition comprising the compound of claim 1, to the subject.
 19. The method of claim 18, wherein the TDP-43 proteinopathy, neurodegenerative or neuromuscular disease is amyotrophic lateral sclerosis, frontotemporal lobar degeneration, age-related macular degeneration, or Alzheimer's disease.
 20. A method of inhibiting expression of FNIP1 and/or FNIP2 in cells or tissues, comprising administering the compound of claim 1, or a pharmaceutical composition comprising the compound of claim 1, to the subject, such that the expression of FNIP1 and/or FNIP2 is inhibited. 